Optical imaging system and methods for using the same

ABSTRACT

Aspects of the present disclosure include methods and systems for assaying a sample for an analyte. Methods according to certain embodiments include illuminating a sample with a slit-shaped beam of light, detecting light transmitted through the sample, determining absorbance of the transmitted light at one or more wavelengths and calculating concentration of the analyte based on the absorbance to assay the sample for the analyte. Systems for practicing the subject methods are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S.Provisional Patent Application Ser. No. 61/903,804, filed on Nov. 13,2013, and U.S. Provisional Patent Application Ser. No. 61/949,833 filedon Mar. 7, 2014, the disclosures of which applications are incorporatedherein by reference.

INTRODUCTION

The characterization of analytes in biological fluids has become anintegral part of medical diagnoses and assessments of overall health andwellness of a patient. In particular, analyte detection in physiologicalfluids, e.g., blood or blood derived products is of ever increasingimportance where the results may play a prominent role in the treatmentprotocol of a patient in a variety of disease conditions. In response tothis growing importance of analyte detection, a variety of analytedetection protocols and devices for laboratory, clinical and at-home usehave been developed.

For example, patients having abnormal levels of hemoglobin often sufferfrom various conditions including anemia, sickle cell anemia, loss ofblood, nutritional deficiency, bone marrow problems and disorders,including polycythemia rubra vera, dehydration, lung disease, certaintumors, and drug abuse, including abuse of the drug erythropoietin.Specific treatment of these conditions often depends on the duration andlevel of hemoglobin abnormality. Therefore, being able to rapidly andaccurately determine the concentration of hemoglobin in the blood of apatient would substantially help in diagnosing and managing conditionsin a patient which arise due to abnormal levels of hemoglobin.

SUMMARY

Aspects of the present disclosure include methods for assaying a samplefor an analyte. Methods according to certain embodiments includeilluminating a sample in a sample chamber with a light source through aslit projection module, detecting light transmitted through the sampleand calculating absorbance of the detected light at one or morewavelengths to assay the sample for the analyte. A slit projectionmodule having a slit that narrows a beam of light from the light sourcecoupled to a focusing lens to focuses light from the slit, amicrocartridge device having a sample chamber for assaying the sampleand imaging systems having a light source, a slit projection module, anobjective lens for focusing light transmitted through the sample and adetector for detecting one or more wavelengths of the transmitted lightsuitable for practicing the subject methods are also described.

As summarized above, aspects of the present disclosure include a methodof assaying a sample for an analyte where the method includes the stepsof illuminating a sample in a sample chamber with a light source througha slit projection module, detecting light transmitted through the sampleand calculating the absorbance of the detected light at one or morewavelengths to assay the sample for the analyte.

In some embodiments, the sample is illuminated with one or more broadspectrum light sources. The sample may be illuminated with light usingone or more visible or near infrared light sources, in certaininstances, with wavelengths which range from 500 nm to 850 nm. Forexample, the sample may be illuminated with two broad spectrum lightsources where the sample is illuminated with first broad spectrum lightsource having a wavelength range from 500 nm to 700 nm and a secondbroad spectrum light source having a wavelength range from 700 nm to 850nm. In certain embodiments, the one or more broad spectrum light sourceshave an irradiation profile with emission peaks at about 450 nm, 550 nmand 830 nm.

In embodiments, the sample is illuminated with the broad spectrum lightsource through a slit projection module having a slit that narrows abeam of light coupled to a focusing lens to focused narrowed light fromthe light source. The slit projection module, when illuminated projectsa beam of light in the shape of a slit onto the sample chamber. In someembodiments, the sample chamber is illuminated by moving the samplechamber across the slit-shaped beam. In other embodiments, the samplechamber is illuminated by moving the slit-shaped projection module alongthe length of the sample chamber.

In some instances, the slit projection module narrows the beam of lightsuch that length of the slit-shaped beam projection is less than thewidth of the sample chamber. In other instances, the slit projectionmodule narrows the beam of light such that the length of the slit-shapedbeam projection is greater than the width of the sample chamber. In yetother instances, the slit projection module narrows the beam of lightsuch that the length of the slit-shaped beam projection is substantiallythe same as the width of the sample chamber. In these embodiments, theslit projection module narrows the beam of light such that theslit-shaped beam projection has a length of from about 2.5 mm to about3.5 mm, such as about 3 mm. In some embodiments, the slit projectionmodule is configured to project a light beam in the shape of a slithaving a width of from about 25 μm to about 75 μm, such as for exampleabout 50 μm.

In some embodiments, the sample is illuminated by moving themicrofluidic chamber containing sample across the slit-shaped beamprojection along 75% or more of the length of the microfluidic chamber.In certain instances, the sample is illuminated by moving the length ofthe sample chamber along the slit-shaped beam projection. In someinstances, the method includes moving the length of the microfluidicchamber along the slit-shaped beam projection in discrete increments,such as for example in 1 mm or greater increments, such as 2 mm orgreater increments and including 5 mm or greater increments. In otherinstances, methods also include continuously moving the length of thesample chamber along the slit-shaped beam projection. In someembodiments, the absorbance of light is measured continuously as thelength of the sample chamber is moved along the slit.

In other embodiments, the sample is illuminated by moving theslit-projection module in a manner sufficient to displace theslit-shaped beam projection along 75% or more of the length of themicrofluidic chamber. In certain instances, the sample is illuminated bymoving the slit-projection module in a manner sufficient to displace theslit-shaped beam projection along the length of the sample chamber. Insome instances, the method includes moving the slit-projection module ina manner sufficient to displace the slit-shaped beam projection alongthe length of the sample chamber in discrete increments, such as forexample in 1 mm or greater increments, such as 2 mm or greaterincrements and including 5 mm or greater increments. In other instances,methods includes moving the slit-projection module in a mannersufficient to continuously move the slit-shaped beam projection alongthe length of the sample chamber. In some embodiments, the absorbance oflight is measured continuously.

In some embodiments, detecting light transmitted through the sampleincludes spatially separating wavelengths of the transmitted light. Incertain instances, spatially separating wavelengths of the transmittedlight includes using a diffraction grating. In certain embodiments,detecting light transmitted through the sample includes projecting anon-diffracted image of the slit on the detector, such as for examplefor use in calibrating the detector.

In embodiments, methods also include calculating absorbance of thedetected light at one or more wavelengths to assay the sample for theanalyte. For example, the absorbance of the detected light may becalculated at two different wavelengths. In certain instances, to assaythe sample for the analyte the absorbance of transmitted light iscalculated at a wavelength between 500 nm and 600 nm, such as at 548 nm.In other instances, to assay the sample for the analyte the absorbanceof transmitted light is calculated at a wavelength between 600 nm and700 nm, such as 650 nm and such as at 675 nm. In yet other instances, toassay the sample for the analyte a first absorbance of transmitted lightis calculated at a wavelength between 500 nm and 600 nm and a secondabsorbance is calculated at a wavelength between 600 nm and 700 nm, suchas calculating absorbance of transmitted light at 548 nm and at 675 nm.In still another instance, to assay the sample for the analyte a firstabsorbance of transmitted light is calculated at a wavelength between500 nm and 600 nm and a second absorbance is calculated at a wavelengthbetween 600 nm and 700 nm, such as calculating absorbance of transmittedlight at 548 nm and at 650 nm.

Aspects of the present disclosure also include systems for practicingthe subject methods. Systems, according to certain embodiments, includea light source for illuminating a sample chamber and a slit projectionmodule which contains a slit that narrows the beam of light from thelight source and a focusing lens for focusing the light narrowed by theslit to provide a slit-shaped beam projection at the sample chamber.Systems also include an objective lens for focusing light transmittedthrough the sample and a detector for detecting one or more wavelengthsof light transmitted through the sample.

In some embodiments, systems include one or more broad spectrum lightsources. The broad spectrum light sources, in certain instances, includeone or more visible or near infrared light sources, such as withwavelengths which range from 500 nm to 850 nm. For example, the firstbroad spectrum light source may have emission wavelengths ranging from500 nm to 700 nm and the second broad spectrum light source havingemission wavelengths ranging from 700 nm to 850 nm. In certainembodiments, the one or more broad spectrum light sources have anirradiation profile with emission peaks at about 450 nm, 550 nm and 830nm.

Systems also include a slit projection module having a slit that narrowsa beam of light from the light source and a focusing lens which focusesthe narrowed beam of light to provide a projection of the beam of lightin the shape of a slit. The slit projection module may be configuredsuch that the length of the slit-shaped beam projection is orthogonal tothe width of the sample chamber. The width of the light beam projectedin the shape of a slit may vary, such as ranging from 75 μm to 125 μm,including 100 μm. The length of the light beam projected in the shape ofa slit may also vary, ranging from 2 mm to 3 mm, such as 2.5 mm. Theslit projection module may be configured to project a beam of light inthe shape of a slit which is greater than the width of the samplechamber. Alternatively, the slit projection module may be configured toproject a beam of light in the shape of a slit which is equal to thewidth of the sample chamber. Likewise, the slit projection module may beconfigured to project a beam of light in the shape of slit which is lessthan the width of the sample chamber.

In some instances, the slit projection module also includes an opticaladjustment protocol. By “optical adjustment” is meant that the beam oflight in the shape of a slit may be changed as desired, such as toincrease or decrease the dimensions or to enhance the optical resolutionof the slit shaped beam. In some instances, optical adjustment is amagnification protocol configured to increase the width of the slit,such as by 5% or greater, such as by 10% or greater, such as by 25% orgreater, such as by 50% or greater and including increasing the width ofthe slit shaped beam by 75% or greater. In other instances, opticaladjustment is a de-magnification protocol configured to decrease thewidth of the slit, such as by 5% or greater, such as by 10% or greater,such as by 25% or greater, such as by 50% or greater and includingdecreasing the width of the slit shaped beam by 75% or greater. Incertain embodiments, optical adjustment is an enhanced resolutionprotocol configured to improve the resolution of the slit shaped beam,such as by 5% or greater, such as by 10% or greater, such as by 25% orgreater, such as by 50% or greater and including enhancing theresolution of the slit shaped beam by 75% or greater. The slit shapedbeam may be adjusted with any convenient optical adjustment protocol,including but not limited to lens, mirrors, pinholes, slits, andcombinations thereof. In certain embodiments, the slit projection moduleincludes a focusing lens coupled to the slit to focus the light narrowedby the slit. The focusing lens, for example may be a de-magnifying lens,such as having a magnification ratio from about 0.5 to 0.75. Forinstance, the de-magnifying lens may be a doublet achromaticde-magnifying lens having a magnification ratio of about 0.6.

Systems according to some embodiments also include an objective lens forfocusing light transmitted through the sample chamber. In someinstances, the objective lens is a magnifying lens, such as having amagnification ratio of from 1.5 to 2.5. For example, the objective lensmay be a double achromatic magnifying lens having a magnification ratioof about 1.7.

As described above, the collected light transmitted through the samplemay be spatially separated into distinct wavelengths for detection. Insome embodiments, systems include a diffraction grating for separatinglight into separate wavelengths. In other embodiments, systems mayinclude a plurality of filters for separating light into distinctwavelengths for detection. In yet other embodiments, systems may includea combination of one or more diffraction gratings and a plurality offilters.

Systems also include a detector for detecting transmitted light from thesample. In some embodiments, the detector is a charged coupled device.The detector, in certain instances, is configured to detect lighttransmitted through the sample at wavelengths ranging from 400 nm to 900nm. For example, the detector may be configured to detect a spectrum oftransmitted light from 500 nm to 800 nm.

In embodiments of the present disclosure, systems are configured toprovide a spatial separation resolution of from 5 nm or less, such as 4nm or less, such as 3 nm or less, such as 2 nm or less and including 1nm or less. As such, in some embodiments systems including the slitprojection module, objective lens, diffraction grating and detector fordetecting transmitted light are configured to provide a spatialseparation resolution of from 5 nm or less, such as 4 nm or less, suchas 3 nm or less, such as 2 nm or less and including 1 nm or less. Inother embodiments, systems including the slit projection module,objective lens, filter wheel and detector detecting transmitted lightare configured to provide a spatial resolution of from 5 nm or less,such as 4 nm or less, such as 3 nm or less, such as 2 nm or less andincluding 1 nm or less.

Aspects of the present disclosure also include a slit projection modulefor assaying a sample according to the subject methods. The slitprojection module, in some embodiments, includes a slit that narrows abeam of light from the light source and a focusing lens which focusesthe narrowed beam of light to provide a beam of light in the shape of aslit. The slit projection module may be configured such that the lengthof the slit is orthogonal to the width of the sample chamber. In certaininstances, the slit projection module is configured to project a lightbeam in the shape of a slit illuminating the sample chamber. The widthof the light beam projected in the shape of a slit may vary, such asranging from 75 μm to 125 μm, including 100 μm. The length of the lightbeam projected in the shape of a slit may also vary, ranging from 2 mmto 3 mm, such as 2.5 mm. The slit projection module may be configured toproject a beam of light in the shape of a slit which is greater than thewidth of the sample chamber. Alternatively, the slit projection modulemay be configured to project a beam of light in the shape of a slitwhich is equal to the width of the sample chamber. Likewise, the slitprojection module may be configured to project a beam of light in theshape of slit which is less than the width of the sample chamber. Incertain embodiments, the slit projection module includes a focusing lenscoupled to the slit to focus the light narrowed by the slit. Thefocusing lens, in certain embodiments, is a de-magnifying lens, such ashaving a magnification ratio from about 0.5 to 0.75. For example, thede-magnifying lens may be a doublet achromatic de-magnifying lens havinga magnification ratio of about 0.6.

Methods also include calculating absorbance of the detected light at oneor more wavelengths to assay the sample for the analyte. For example,the absorbance of the detected light may be calculated at two differentwavelengths. In certain instances, to assay the sample for the analytethe absorbance of transmitted light is calculated at a wavelengthbetween 500 nm and 600 nm, such as at 548 nm. In other instances, toassay the sample for the analyte the absorbance of transmitted light iscalculated at a wavelength between 600 nm and 700 nm, such as at 675 nm.In yet other instances, to assay the sample for the analyte a firstabsorbance of transmitted light is calculated at a wavelength between500 nm and 600 nm and a second absorbance is calculated at a wavelengthbetween 600 nm and 700 nm, such as calculating absorbance of transmittedlight at 548 nm and at 675 nm, including calculating absorbance oftransmitted light at 548 nm and at 650 nm.

Aspects of the present disclosure also include a micro-fluidic deviceconfigured to perform an assay of a liquid sample, where the deviceincludes a connected sample application site, and inlet, a capillarychannel sample and a reagent mixing chamber for contacting a sample withone or more reagents. The microfluidic device in certain embodimentsalso includes a blank reference window configured for providing a blankduring absorbance measurement.

BRIEF DESCRIPTION OF THE FIGURES

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 illustrates an example of illuminating a sample chamber with aslit-shaped beam provided by a slit projection module according tocertain embodiments.

FIGS. 2 a and 2 b illustrates an example of configurations of absorbancesystems having a slit projection module for illuminating a samplechamber with a slit-shaped beam according to certain embodiments. FIG. 2a depicts a side view of absorbance systems having a slit projectionmodule. FIG. 2 b depicts a top view of absorbance systems having a slitprojection module.

FIG. 3 illustrates an example of a configuration of absorbance systemshaving a slit-projection module coupled to a fluorescence detectionsystem according to certain embodiments.

FIG. 4 depicts an example of a system of interest according to certainembodiments.

FIG. 5 illustrates an example a microfluidic cartridge having amicrofluidic sample chamber and a reference slit window for providingblank transmittance according to certain embodiments.

FIG. 6 shows an example of a kit having a microfluidic cartridgeaccording to certain embodiments.

FIG. 7 shows an example of a collection of different types of kitsprovided together in a box according to certain embodiments.

FIG. 8 shows a schematic of determining hemoglobin concentrationaccording to certain embodiments.

FIGS. 9 a-c illustrate example hemoglobin absorbance spectra acquired byilluminating a sample chamber through a slit projection module accordingto certain embodiments. FIG. 9 a depicts an absorbance spectrum ofhemoglobin at concentration of 25 g/dL in whole blood. FIG. 9 b depictsan absorbance spectrum of hemoglobin at concentration of 7 g/dL in wholeblood. FIG. 9 c depicts a plot of hemoglobin concentration andabsorbance at 569 nm.

FIG. 10 illustrates a comparison of hemoglobin measurement in wholeblood with methods according to certain embodiments and a hematologyanalyzer.

DETAILED DESCRIPTION

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, the present disclosure provides methods forassaying a sample for one or more analytes. In further describingembodiments of the disclosure, methods for assaying a sample for ananalyte are first described in greater detail. Next, systems suitablefor practicing the subject methods to assay the sample for the analyteare described. Microcartridges, computer controlled systems and kits arealso provided.

Methods for Assaying a Sample for an Analyte

As summarized above, aspects of the present disclosure include methodsfor assaying a sample for one or more analytes. The term “assaying” isused herein in its conventional sense to refer to qualitativelyassessing or quantitatively measuring the presence or amount of a targetanalyte species. In certain embodiments, methods include assaying asample for hemoglobin.

A variety of different samples may be assayed according to methods ofthe invention, e.g., as described herein. In some instances, the sampleis a biological sample. The term “biological sample” is used in itsconventional sense to refer to a whole organism, plant, fungi or asubset of animal tissues, cells or component parts which may in certaininstances be found in blood, mucus, lymphatic fluid, synovial fluid,cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid,amniotic cord blood, urine, vaginal fluid and semen. As such, a“biological sample” refers to both the native organism or a subset ofits tissues as well as to a homogenate, lysate or extract prepared fromthe organism or a subset of its tissues, including but not limited to,for example, plasma, serum, spinal fluid, lymph fluid, sections of theskin, respiratory, gastrointestinal, cardiovascular, and genitourinarytracts, tears, saliva, milk, blood cells, tumors, organs. Biologicalsamples may be any type of organismic tissue, including both healthy anddiseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certainembodiments, the biological sample is a liquid sample, such as blood orderivative thereof, e.g., plasma, tears, urine, semen, etc., where insome instances the sample is a blood sample, including whole blood, suchas blood obtained from venipuncture or fingerstick (where the blood mayor may not be combined with any reagents prior to assay, such aspreservatives, anticoagulants, etc.).

In certain embodiments the source of the sample is a “mammal” or“mammalian”, where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some instances,the subjects are humans. The methods may be applied to samples obtainedfrom human subjects of both genders and at any stage of development(i.e., neonates, infant, juvenile, adolescent, adult), where in certainembodiments the human subject is a juvenile, adolescent or adult. Whilethe present invention may be applied to samples from a human subject, itis to be understood that the methods may also be carried-out on samplesfrom other animal subjects (that is, in “non-human subjects”) such as,but not limited to, birds, mice, rats, dogs, cats, livestock and horses.

In embodiments, the amount of sample assayed in the subject methods mayvary, for example, ranging from 0.01 μL to 1000 μL, such as from 0.05μl_(—) to 900 μL, such as from 0.1 μl_(—) to 800 μL, such as from 0.5μl_(—) to 700 μL, such as from 1 μl_(—) to 600 μL, such as from 2.5μl_(—) to 500 μL, such as from 5 μl_(—) to 400 μL, such as from 7.5μl_(—) to 300 μl_(—) and including from 10 μl_(—) to 200 μl_(—) ofsample.

In some embodiments, the biological sample is a specimen that has beenpreloaded into a container (e.g., blender cup, vortex microtube,sonicator vessel, etc.) and stored for a predetermined period of timebefore the biological sample is assayed. For example, the biologicalsample may be preloaded into a microfluidic cartridge, as described ingreater detail below, for a period of time before the biological sampleis assayed according to the subject methods. The amount of time thebiological sample is stored following preloading into the containerbefore assaying the biological sample may vary, such as 0.1 hours ormore, such as 0.5 hours or more, such as 1 hour or more, such as 2 hoursor more, such as 4 hours or more, such as 8 hours or more, such as 16hours or more, such as 24 hours or more, such as 48 hours or more, suchas 72 hours or more, such as 96 hours or more, such as 120 hours ormore, such as 144 hours or more, such as 168 hours or more and includingpreloading the biological sample into the container 240 hours or morebefore assaying the biological sample or may range such as from 0.1hours to 240 hours before assaying the biological sample, such as from0.5 hours to 216 hours, such as from 1 hour to 192 hours and includingfrom 5 hours to 168 hours before assaying the biological sample. Forexample, the biological sample may be preloaded into a container (e.g.,microfluidic cartridge) configured for use with a system (as describedbelow) for assaying the sample at a remote location (e.g., at home usingan at-home kit or in a physician's office) and sent to a laboratory forassaying in accordance with the subject methods. By “remote location” ismeant a location other than the location at which the sample iscontained and preloaded into the container. For example, a remotelocation could be another location (e.g. office, lab, etc.) in the samecity, another location in a different city, another location in adifferent state, another location in a different country, etc., relativeto the location of the processing device, e.g., as described in greaterdetail below. In some instances, two locations are remote from oneanother if they are separated from each other by a distance of 10 m ormore, such as 50 m or more, including 100 m or more, e.g., 500 m ormore, 1000 m or more, 10,000 m or more, etc.

In practicing methods according to certain embodiments, a sample in asample chamber is illuminated with a light source through a slitprojection module, detecting light transmitted through the sample andcalculating absorbance of the detected light at one or more wavelengthsto assay the sample for the analyte. Depending on the target analyte,the sample may be illuminated with one or more sources of light. In someembodiments, the sample is illuminated with one or more broadband lightsources. The term “broadband” is used herein in its conventional senseto refer to a light source which emits light having a broad range ofwavelengths, such as for example, spanning 50 nm or more, such as 100 nmor more, such as 150 nm or more, such as 200 nm or more, such as 250 nmor more, such as 300 nm or more, such as 350 nm or more, such as 400 nmor more and including spanning 500 nm or more. For example, one suitablebroadband light source emits light having wavelengths from 400 nm to 700nm. Another example of a suitable broadband light source includes alight source that emits light having wavelengths from 500 nm to 700 nm.Any convenient broadband light source protocol may be employed, such asa halogen lamp, deuterium arc lamp, xenon arc lamp, stabilizedfiber-coupled broadband light source, a broadband LED with continuousspectrum, superluminescent emitting diode, semiconductor light emittingdiode, wide spectrum LED white light source, an multi-LED integratedwhite light source, among other broadband light sources or anycombination thereof.

In other embodiments, the sample is illuminated with one or more narrowband light sources emitting a particular wavelength or narrow range ofwavelengths. The term “narrow band” is used herein in its conventionalsense to refer to a light source which emits light having a narrow rangeof wavelengths, such as for example, 50 nm or less, such as 40 nm orless, such as 30 nm or less, such as 25 nm or less, such as 20 nm orless, such as 15 nm or less, such as 10 nm or less, such as 5 nm orless, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Any convenientnarrow band light source protocol may be employed, such as a narrowwavelength LED, laser diode or a broadband light source coupled to oneor more optical bandpass filters, diffraction gratings, monochromatorsor any combination thereof.

Depending on the analyte being assayed as well as interferents presentthe biological sample, the biological sample may be illuminated usingone or more light sources, such as two or more light sources, such asthree or more light sources, such as four or more light sources, such asfive or more light sources and including ten or more light sources. Anycombination of light sources may be used, as desired. For example, wheretwo lights sources are employed, a first light source may be a broadbandwhite light source (e.g., broadband white light LED) and second lightsource may be a broadband near-infrared light source (e.g., broadbandnear-IR LED). In other instances, where two light sources are employed,a first light source may be a broadband white light source (e.g.,broadband white light LED) and the second light source may be a narrowspectra light source (e.g., a narrow band visible light or near-IR LED).In yet other instances, the light source is an plurality of narrow bandlight sources each emitting specific wavelengths, such as an array oftwo or more LEDs, such as an array of three or more LEDs, such as anarray of five or more LEDs, including an array of ten or more LEDs.

Where more than one light source is employed, the sample may beilluminated with the light sources simultaneously or sequentially, or acombination thereof. For example, where the sample is illuminated withtwo light sources, the subject methods may include simultaneouslyilluminating the sample with both light sources. In other embodiments,the sample may be sequentially illuminated by two light sources. Wherethe sample is sequentially illuminated with two or more light sources,the time each light source illuminates the same may independently be0.001 seconds or more, such as 0.01 seconds or more, such as 0.1 secondsor more, such as 1 second or more, such as 5 seconds or more, such as 10seconds or more, such as 30 seconds or more and including 60 seconds ormore. In embodiments where the sample is sequentially illuminated by twoor more light sources, the duration the sample is illuminated by eachlight source may be the same or different.

The time period between illumination by each light source may also vary,as desired, being separated independently by a delay of 1 second ormore, such as 5 seconds or more, such as by 10 seconds or more, such asby 15 seconds or more, such as by 30 seconds or more and including by 60seconds or more. In embodiments where the sample is sequentiallyilluminated by more than two (i.e., three or more) light sources, thedelay between illumination by each light source may be the same ordifferent.

Depending on the assay protocol, illumination of the sample may becontinuous or in discrete intervals. For example, in some embodiments,the sample may be illuminated continuously throughout the entire timethe sample is being assayed. Where the light includes two or more lightsources, the sample may be continuously illuminated by all of the lightsources simultaneously. In other instances, the sample is continuouslyilluminated with each light source sequentially. In other embodiments,the sample may be illuminated in regular intervals, such as illuminatingthe sample every 0.001 microseconds, every 0.01 microseconds, every 0.1microseconds, every 1 microsecond, every 10 microseconds, every 100microseconds and including every 1000 microseconds.

The sample may be illuminated with the light source one or more times atany given measurement period, such as 2 or more times, such as 3 or moretimes, including 5 or more times at each measurement period.

As described in greater detail below, the light source for illuminatingthe sample may emit a spectrum of light having wavelengths ranging from400 nm to 900 nm, such as from 450 nm to 850 nm, such as from 500 nm to800 nm, such as from 550 nm to 750 nm and including from 600 nm to 700nm. In some embodiments, the sample is illuminated with a single broadband light source emitting light with wavelengths from 400 nm to 900 nm.In other embodiments, the sample is illuminated with light withwavelengths from 400 nm to 900 nm using a plurality of light sources.For example, the sample may be illuminated with by a plurality of narrowband light sources each independently emitting light having wavelengthsin the range of 400 nm to 900 nm.

In certain embodiments, the sample is illuminated with two broadbandlight sources emitting light with wavelengths from 400 nm to 900 nm. Forexample, the light sources may be white light LED emitting light havingwavelengths ranging from 400 nm to 700 nm and a near-infrared LEDemitting light having wavelengths ranging from 700 nm to 900 nm.Depending on the type of light source, as described above, theirradiation profile of each light source may vary, having any number ofemission peaks. In certain instances, the sample is illuminated with awhite light LED emitting light having wavelengths ranging from 400 nm to700 nm and having emission peaks at about 450 nm and 550 nm and anear-infrared LED emitting light having wavelengths ranging from 700 nmto 900 nm and having an emission peak at about 830 nm.

In other embodiments, the sample is illuminated with by a plurality ofnarrow band lamps or LEDs each independently emitting specificwavelengths of light in the range of 400 nm to 900 nm. In one example,the narrow band light source is one or more monochromatic LEDs emittinglight in the range of 500 nm to 700 nm, such as at 504 nm, 506 nm, 514nm, 532 nm, 543 nm, 548 nm, 550 nm, 561 nm, 568 nm, 579 nm, 580 nm, 585nm, 586 nm or any combination thereof. In another example, the narrowband light source is one or more narrow band lamps emitting light in therange of 500 nm to 700 nm, such as a narrow band cadmium lamp, cesiumlamp, helium lamp, mercury lamp, mercury-cadmium lamp, potassium lamp,sodium lamp, neon lamp, zinc lamp or any combination thereof.

In embodiments of the present disclosure the sample is illuminated witha slit of illuminating light. The slit of illuminating light, e.g., asdescribed in greater detail below, may be produced using any convenientprotocol. In some embodiments, the slit of illuminating light isproduced using a slit projection module, which includes a slit that mayor may not be optically coupled to one or more additional components,e.g., one or more lenses. For example, in some instances the slit ofilluminating light is produced from one or more light sources through aslit projection module having a slit that narrows a beam of lightcoupled to a focusing lens to focus the narrowed beam of light from thelight source. The slit projection module narrows the beam of light andproduces a beam of light in the shape of a slit projected onto thesample chamber. During interrogation of the sample, the sample chamber,the slit projection module or both the sample chamber and slitprojection module may be moved (if desired) to displace the slit-shapedbeam of light across the sample chamber.

As described above, the slit projection module is configured to providea slit-shaped beam having a length and width, where the length and widthof the illuminating slit may vary. The slit projection module includes aslit having an aperture configured to narrow the beam of light from theone or more light sources and a focusing lens coupled to the slit forfocusing the light passing through the slit aperture. The slit aperturemay be any convenient shape, including but not limited to an oval,rectangle or other polygon. In certain embodiments, the slit aperture isrectangular. Depending on the desired dimensions of slit-shaped beamprovided by the light source, as described above, the dimensions of theslit aperture may vary, having a length which ranges from 01 mm to 10mm, such as from 1.25 mm to 9.5 mm, such as from 1.5 mm to 9 mm, such asfrom 2 mm to 8 mm, such as from 2.5 mm to 7 mm, such as from 3 mm to 6mm and including from 3.5 mm to 5 mm. The width of the slit aperture mayrange from 1 μm to 250 μm, such as from 2 μm to 225 μm, such as from 5μm to 200 μm, such as from 10 μm to 150 μm, and including from 15 μm to125 μm, for example a slit having an aperture width of 100 μm.

The light beam narrowed by the slit may, where desired, be focused usinga focusing lens coupled to the slit. In some embodiments, the narrowedlight beam is focused through a de-magnifying lens having amagnification ratio ranging from 0.1 to 0.95, such as a magnificationratio of from 0.2 to 0.9, such as a magnification ratio of from 0.3 to0.85, such as a magnification ratio of from 0.35 to 0.8, such as amagnification ratio of from 0.5 to 0.75 and including focusing thenarrowed light beam through a de-magnifying lens having a magnificationratio of from 0.55 to 0.7, for example a magnification ratio of 0.6. Forexample, the narrowed light beam is, in certain instances, focusedthrough a double achromatic de-magnifying lens having a magnificationratio of about 0.6.

As described in greater detail below, the slit projection module may beconfigured to provide a slit-shaped beam having a length and width whichvaries. In some embodiments, the slit projection module is configured toprovide a slit-shaped beam having a length which ranges from 1 mm to 5mm, such as from 1.5 mm to 4.5 mm, such as from 2 mm to 4 mm, such asfrom 2.5 mm to 3.5 mm and including a slit-shaped beam having a lengthof 3 mm. In these embodiments, the slit projection module is configuredto provide a slit-shaped beam having a width which ranges from 10 μm to100 μm, such as from 15 μm to 95 μm, such as from 20 μm to 90 μm, suchas from 25 μm to 85 μm, such as from 30 μm to 80 μm, such as from 35 μmto 75 μm, such as from 40 μm to 70 μm, such as from 45 μm to 65 μm, andincluding from 50 μm to 60 μm.

In some embodiments, the slit projection module is configured to providea slit-shaped beam of light where the length of the slit-shaped beam isorthogonal to the length of a sample chamber being assayed. In otherwords, the length of the slit-shaped beam is positioned along the widthof the sample chamber being assayed. Depending on the size of the samplechamber (as described in greater detail below) and slit-shaped beamprojection, the slit-shaped beam may illuminate 50% or more of the widthof the sample chamber, such as 55% or more, such as 60% or more, such as65% or more, such as 70% or more, such as 75% or more, such as 80% ormore, such as 85% or more, such as 90% or more, such as 95% or more,such as 97% or more and including illuminating 99% or more of the widthof the sample chamber. In certain instances, the slit-shaped beamprojection has a length which is substantially the same as width of thesample chamber. In other embodiments, the slit-projection module isconfigured to provide a slit-shaped beam projection which has a lengththat is greater than the width of the sample chamber. For example, theslit-shaped beam of light may have a length which is 1% or greater thanthe width of the sample chamber, such as 2% or greater, such as 5% orgreater, such as 10% or greater, such as 15% or greater, such as 20% orgreater and including a length which is 25% greater than the width ofthe sample chamber. In yet other embodiments, the slit-projection moduleis configured to provide a slit-shaped beam projection which has alength that is less than the width of the sample chamber. For example,the slit-shaped beam of light may have a length is which 1% or greaterless than the width of the sample chamber, such as a length which is 2%or greater less than the width of the sample chamber, such as a lengthwhich is 5% or greater less than the width of the sample chamber, suchas a length which is 10% or greater less than the width of the samplechamber, such as a length which is 15% or greater less than the width ofthe sample chamber, such as a length which is 20% or greater less thanthe width of the sample chamber and including a length which is 25% orgreater less than the width of the sample chamber.

During interrogation of the sample, the sample chamber, the slitprojection module or both the sample chamber and slit projection modulemay be moved (if desired) to displace the slit-shaped beam of lightacross the sample chamber. The term “move” refers to displacementbetween slit projection module and the sample chamber such that theslit-shaped beam of light projected onto the sample chamber changesposition with time along the sample chamber during assay of the sample.In some embodiments, the sample chamber is moved while theslit-projection module is maintained in a stationary position in orderto laterally displace the slit-shaped beam of light along the samplechamber during interrogation. In other embodiments, the slit-projectionmodule is moved and the sample chamber is maintained in a stationaryposition in order to laterally displace the slit-shaped beam of lightalong the sample chamber during interrogation. In yet other embodiments,both the slit projection module and the sample are moved in order tolaterally displace the slit-shaped beam of light along the samplechamber during interrogation.

In embodiments, the sample chamber or the slit-projection module may bemoved such that the slit-shaped beam of light is displaced laterallyrelative to the sample chamber in any direction, where in some instancesthe length of the slit-shaped beam remains orthogonal to the length ofthe sample chamber. In some instances, the sample chamber or theslit-projection module is moved such that the slit-shaped beam of lightis displaced from a distal end to a proximal end of the sample chamber.In other instances, the sample chamber or the slit-projection module ismoved such that the slit-shaped beam of light is displaced from aproximal end to a distal end of the sample chamber. The sample chamberor the slit-projection module may be configured to move such that theslit-shaped beam of light is displaced along all or a portion of thelength of the sample chamber to interrogate the sample. In someembodiments, the slit-shaped beam of light is displaced along 50% ormore of the sample chamber, such as 55% or more, such as 60% or more,such as 65% or more, such as 70% or more, such as 75% or more, such as80% or more, such as 85% or more, such as 90% or more, such as 95% ormore, such as 97% or more and including along 99% or more of the lengthof the sample chamber. In certain instances, the slit-shaped beam isdisplaced along substantially the entire length of the sample chamber.

In certain embodiments, sample chamber or slit-projection module ismoved so that the slit-shaped beam light is displaced in aback-and-forth motion relative to the sample chamber, such as movingfrom a distal end to a proximal end of the sample chamber and back fromthe proximal end to the distal end of the sample chamber. In otherinstances, the sample chamber or slit-projection module is moved so thatthe slit-shaped beam of light is displaced relative to the samplechamber from the proximal end to distal end of the sample chamber andback from the distal end to the proximal end of the sample chamber. Thesample chamber or slit-projection module is moved so that theslit-shaped beam of light is, in certain instances, displaced in aback-and-forth motion along only a portion of the sample chamber. Forexample, the sample chamber or slit-projection module is moved so thatthe slit-shaped beam may be displaced in a back-and-forth motion along99% or less of the sample chamber, such as 95% or less, such as 90% orless, such as 85% or less, such as 80% or less, such as 75% or less,such as 70% or less, such as 65% or more, including moving the samplechamber or slit-projection module is moved so that the slit-shaped beamis displaced in a back-and-forth motion along 50% or less of the lengthof the sample chamber.

Where the slit-shaped beam of light is moved in a back-and-forth motion,the movement of the sample chamber or slit-projection module may berepeated one or more times during any given interrogation period, suchas 2 or more times, such as 5 or more times, such as 10 or more times,such as 15 or more times and including 25 or more times during eachinterrogation period.

The amplitude of displacement of the slit-shaped beam of light along thesample chamber during interrogation may vary. By “amplitude ofdisplacement” or “total displacement” is meant the sum total of distancetraversed by the slit-shaped beam of light along the sample chamber. Inone example, where the slit-shaped beam of light is moved from aproximal end to a distal end of a 60 mm sample chamber, the totaldisplacement of the slit-shaped beam of light is 60 mm. In anotherexample, where the slit-shaped beam is moved from a distal end to aproximal end of a 60 mm sample chamber, the total displacement of theslit-shaped beam of light is 60 mm. In yet another example, where theslit-shaped beam is moved in a back-and-forth motion from the proximalend to a distal end and back from the distal end to the proximal end ofa 60 mm sample chamber, the total displacement is 120 mm. In stillanother example, where the slit-shaped beam is moved in a back-and-forthmotion (e.g., from proximal end to distal end and back from distal endto proximal end) along 50% of a 60 mm sample chamber, the totaldisplacement is 60 mm. In still another example, where the slit-shapedbeam is moved in a back-and-forth motion (e.g., from distal end toproximal end and back from proximal end to distal end) along 50% of a 60mm sample chamber and repeated 5 times, the total displacement is 300mm.

In embodiments of the present disclosure, the total displacement varies,ranging from 0.1 mm to 1000 mm, such as from 0.2 mm to 950 mm, such asfrom 0.3 mm to 900 mm, such as from 0.4 mm to 850 mm, such as from 0.5mm to 800 mm, such as from 0.6 mm to 750 mm, such as from 0.7 mm to 700mm, such as from 0.8 mm to 650 mm, such as from 0.9 mm to 600 mm, suchas from 1 mm to 550 mm, such as from 1.25 mm to 500 mm, such as from 1.5mm to 450 mm, such as from 1.75 mm to 400 mm, such as from 2 mm to 300mm, such as from 2.5 mm to 250 mm and including from 3 mm to 200 mm.

The slit-shaped beam of light may be moved along the sample chamber at arate which varies, such as at a rate of 0.001 mm/second or more, such asat a rate of 0.005 mm/second or more, such as at a rate of 0.01mm/second or more, such as at a rate of 0.05 mm/second or more, such asat a rate of 0.1 mm/second or more, such as at a rate of 0.5 mm/secondor more, such as at a rate of 1 mm/second or more, such as at a rate of1.5 mm/second or more, such as at a rate of 2 mm/second or more, such asat a rate of 2.5 mm/second or more, such as at a rate of 3 mm/second,such as at a rate of 5 mm/second or more, such as at a rate of 10mm/second or more, such as at a rate of 20 mm/second or more, such as ata rate of 30 mm/second or more, such as at a rate of 40 mm/second ormore and including moving the slit-shaped beam along the sample chamberat a rate of 60 mm/second or more. Where the slit-shaped beam is movedalong the sample chamber in a back-and-forth motion, the rate may be 1back-and-forth cycle per minute or more, such as 2 cycles per minute ormore, such as 3 cycles per minute or more, such as 4 cycles per minuteor more, such as 5 cycles per minute or more, such as 10 cycles perminute or more, such as 15 cycles per minute or more, such as 20 cyclesper minute or more, such as 30 cycles per minute or more, such as 45cycles per minute or more and including 60 back-and-forth cycles perminute or more.

Movement of the sample chamber or slit-projection module to displace theslit-shaped beam along the sample chamber may be continuous or indiscrete increments. In some embodiments, the sample chamber orslit-projection module is moved so that the slit-shaped beam of light isdisplaced along the sample chamber continuously throughout the entiretime the sample is being assayed, such as at a rate of 0.001 mm/secondor more, such as at a rate of 0.005 mm/second or more, such as at a rateof 0.01 mm/second or more, such as at a rate of 0.05 mm/second or more,such as at a rate of 0.1 mm/second or more, such as at a rate of 0.5mm/second or more, such as at a rate of 1 mm/second or more, such as ata rate of 1.5 mm/second or more, such as at a rate of 2 mm/second ormore, such as at a rate of 2.5 mm/second or more, such as at a rate of 3mm/second, such as at a rate of 5 mm/second or more, such as at a rateof 10 mm/second or more, such as at a rate of 20 mm/second or more, suchas at a rate of 30 mm/second or more, such as at a rate of 40 mm/secondor more and including moving the sample chamber or slit-projectionmodule so that the slit-shaped beam of light is continuously displacedrelative to the sample chamber at a rate of 60 mm/second or more.

In other embodiments, the sample chamber or slit-projection module ismoved so that the slit-shaped beam is displaced relative to the samplechamber in discrete increments. In these embodiments, the slit-shapedbeam of light is, in some instances, displaced relative to the samplechamber in regular increments, such as at 0.01 mm increments, 0.05 mmincrements, 0.1 mm increments, 0.2 mm increments, 0.3 mm increments, 0.5mm increments, 0.75 mm increments, 1 mm increments, 2.5 mm increments, 5mm increments, 7.5 mm increments, 10 mm increments, 15 mm increments, 20mm increments or some other increment. In other instances, theslit-shaped beam of light is displaced relative to the sample chamber inrandom increments ranging from 0.01 mm to 20 mm increments, such as from0.1 mm to 17.5 mm, such as from 0.5 mm to 15 mm increments, includingrandom increments ranging from 1 mm to 10 mm increments.

As described above, in some embodiments the sample chamber issequentially illuminated with a plurality of light sources. Where thesample chamber is sequentially illuminated with more than one lightsource, each light source independently provides a slit-shaped beam oflight which is displaced relative to the sample chamber. The movement ofeach slit-shaped light beam of light relative to the sample chamberprovided by the plurality of light sources may be the same or different,as described above. For example, where the sample chamber issequentially illuminated with two light sources (e.g., a broadband whitelight LED and a near-IR LED), a first slit-shaped beam produced by thefirst light source (e.g., broadband white light LED) interrogates thesample chamber sequentially with a second slit-shaped beam produced bythe second light source (e.g., near-IR LED). In other words, in theseembodiments at least two slit-shaped beams are provided to interrogatethe sample chamber. For example, in some embodiments a first lightsource providing a first slit-shaped beam may be moved along the samplechamber in a back-and-forth motion while a second light source providinga second slit-shaped beam may be moved along the sample chamber in onlya single direction. In other embodiments, a first light source providinga first slit-shaped beam may be moved along the sample chamber in aback-and-forth motion for a plurality of cycles while the second lightsource providing a second slit-shaped beam may be moved in aback-and-forth motion for a single cycle.

FIG. 1 depicts displacement of a slit-shaped beam of light relative to asample chamber. The slit-shaped beam (101) provided by a slit projectionmodule is oriented across the width of the sample chamber (102) and thesample chamber is moved laterally (103) across the slit-shaped beam oflight to illuminating all or part of the sample chamber. As desired, thesample chamber or the slit-projection module may be moved so that theslit-shaped beam can be displaced relative to the sample chamber one ormore times or in a back-and-forth motion.

In some embodiments, the light transmitted though the sample chamber iscollected and passed through one or more objective lenses. In certaininstances, light transmitted through the sample chamber is collected andpassed through a magnifying lens with a nominal magnification rangingfrom 1.2 to 2.5, such as a nominal magnification of from 1.3 to 2.4,such as a nominal magnification of from 1.4 to 2.3, such as a nominalmagnification of from 1.5 to 2.2, such as a nominal magnification orfrom 1.6 to 2.1, including passing the transmitted light through amagnifying lens having a nominal magnification of from 1.7 to 2.0, forexample a nominal magnification of 1.7. For example, the transmittedlight is, in certain instances, collected and passed through amagnifying achromatic doublet lens with a nominal magnification of 1.7.Depending on the configuration of the light source, sample chamber anddetector, properties of the objective lens may vary.

For example, the numerical aperture of the subject objective lens mayalso vary, ranging from 0.01 to 1.7, such as from 0.05 to 1.6, such asfrom 0.1 to 1.5, such as from 0.2 to 1.4, such as from 0.3 to 1.3, suchas from 0.4 to 1.2, such as from 0.5 to 1.1 and including a numericalaperture ranging from 0.6 to 1.0. Likewise, the focal length of theobjective lens varies, ranging from 10 mm to 20 mm, such as from 10.5 mmto 19 mm, such as from 11 mm to 18 mm and including from 12 mm to 15 mm.

In some embodiments, the slit-shaped beam projection transmitted throughthe sample chamber is also focused using an autofocus module. Forexample, suitable protocols for focusing the slit-shaped beam projectiontransmitted through the sample chamber may include, but are not limited,to those described in U.S. Pat. No. 6,441,894, filed on Oct. 29, 1999,the disclosure of which is herein incorporated by reference.

Methods according to some embodiments of the present disclosure alsoinclude passing the transmitted light through one or more wavelengthseparators. The term “wavelength separator” is used herein in itsconventional sense to refer to an optical protocol for separatingpolychromatic light into its component wavelengths for detection.Wavelength separation, according to certain embodiments, may includeselectively passing or blocking specific wavelengths or wavelengthranges of the polychromatic light. To separate wavelengths of light, thetransmitted light may be passed through any convenient wavelengthseparating protocol, including but not limited to colored glass,bandpass filters, interference filters, dichroic mirrors, diffractiongratings, monochromators and combinations thereof, among otherwavelength separating protocols.

In some embodiments, methods include separating the light transmittedfrom the sample chamber by passing the transmitted light through one ormore diffraction gratings. Diffraction gratings of interest may include,but are not limited to transmission, dispersive or reflectivediffraction gratings. Suitable spacings of the diffraction grating mayvary depending on the configuration of the light source, slit projectionmodule, sample chamber, objective lens, ranging from 0.01 μm to 10 μm,such as from 0.025 μm to 7.5 μm, such as from 0.5 μm to 5 μm, such asfrom 0.75 μm to 4 μm, such as from 1 μm to 3.5 μm and including from 1.5μm to 3.5 μm. In other embodiments, methods include separating the lighttransmitted from the sample chamber by passing the transmitted lightthrough one or more optical filters, such as one or more bandpassfilters. For example, optical filters of interest may include bandpassfilters having minimum bandwidths ranging from 2 nm to 100 nm, such asfrom 3 nm to 95 nm, such as from 5 nm to 95 nm, such as from 10 nm to 90nm, such as from 12 nm to 85 nm, such as from 15 nm to 80 nm andincluding bandpass filters having minimum bandwidths ranging from 20 nmto 50 nm. In certain instances, methods include passing the transmittedlight from the sample chamber through one or more bandpass filters whichselectively pass wavelengths in the ranges of: 498 nm-510 nm; 500 nm-600nm; 500 nm-520 nm; 540 nm-550 nm; 545 nm-555 nm; 550 nm-570 nm; 550nm-580 nm; 560 nm-590 nm; 575 nm-595 nm; 580 nm-590 nm; 600 nm-700 nm;600 nm-630 nm; 650 nm-750 nm; 750 nm-850 nm; 810 nm-830 nm; 815 nm-825nm or any combination thereof.

For example, in one instance methods include passing the transmittedlight from the sample chamber through one or more bandpass filters whichselectively passes wavelengths ranging from 500 nm-520 nm and from 650nm-750 nm. In another instance, methods include passing the transmittedlight from the sample chamber through one or more bandpass filters whichselectively passes wavelengths ranging from 540 nm-560 nm and from 650nm-750 nm. In yet another instance, methods include passing thetransmitted light from the sample chamber through one or more bandpassfilters which selectively passes wavelengths ranging from 560 nm-590 nmand from 650 nm-750 nm. In still another instance, methods includepassing the transmitted light from the sample chamber through one ormore bandpass filters which selectively passes wavelengths ranging from500 nm-520 nm; 560 nm-590 nm and from 650 nm-750 nm.

In practicing methods according to aspects of the present disclosure,the light transmitted through the sample chamber is measured at one ormore wavelengths. In embodiments, the transmitted light is measured atone or more wavelengths, such as at 5 or more different wavelengths,such as at 10 or more different wavelengths, such as at 25 or moredifferent wavelengths, such as at 50 or more different wavelengths, suchas at 100 or more different wavelengths, such as at 200 or moredifferent wavelengths, such as at 300 or more different wavelengths andincluding measuring the light transmitted through the sample chamber at400 or more different wavelengths.

In some embodiments, measuring light transmitted through the samplechamber includes measuring transmitted light over a range of wavelengths(e.g., 400 nm-800 nm; 495 nm-525 nm; 800 nm-835 nm, etc.). For example,methods may include measuring light transmitted through the samplechamber over one or more of the wavelength ranges of: 400 nm-800 nm; 498nm-510 nm; 500 nm-600 nm; 500 nm-700 nm; 500 nm-575 nm; 500 nm-550 nm;540 nm-550 nm; 545 nm-555 nm; 550 nm-570 nm; 550 nm-580 nm; 560 nm-590nm; 575 nm-595 nm; 580 nm-590 nm; 600 nm-700 nm; 600 nm-630 nm; 650nm-750 nm; 650 nm-830; 750 nm-850 nm; 810 nm-830 nm; 815 nm-825 nm andany combinations thereof. In one instance, methods include measuringtransmitted light over the wavelengths ranging from 400 nm-800 nm. Inanother instance, methods include measuring transmitted light over thewavelengths ranging from 500 nm-520 nm and 650 nm-750 nm. In anotherinstance, methods include measuring transmitted light over wavelengthsranging from 540 nm-560 nm and 650 nm-750 nm. In yet another instance,methods include measuring transmitted light over wavelengths rangingfrom 560 nm-590 nm and 650 nm-750 nm. In still another instance, methodsinclude measuring transmitted light over wavelengths ranging from 500nm-520 nm, 560 nm-590 nm, and 650-750 nm.

Measuring transmitted light over a range of wavelengths, in certaininstances, includes collecting the spectra of the transmitted light overthe range of wavelengths. For example, methods may include collectingthe spectra of light transmitted through the sample chamber over one ormore of the wavelength ranges of: 400 nm-800 nm; 498 nm-510 nm; 500nm-600 nm; 500 nm-700 nm; 500 nm-520 nm; 540 nm-550 nm; 545 nm-555 nm;550 nm-570 nm; 550 nm-580 nm; 560 nm-590 nm; 575 nm-595 nm; 580 nm-590nm; 600 nm-700 nm; 600 nm-630 nm; 650 nm-750 nm; 750 nm-850 nm; 810nm-830 nm; 815 nm-825 nm and any combinations thereof. In one instance,methods include collecting the spectra of transmitted light over thewavelengths ranging from 400 nm-800 nm. In another instance, methodsinclude collecting the spectra of transmitted light over the wavelengthsranging from 500 nm-700 nm.

In certain embodiments, the light transmitted through the sample chamberis detected at one or more specific wavelengths (e.g., 548 nm or 675nm). For example, methods may include detecting the transmitted light at2 or more specific wavelengths, such as at 3 or more specificwavelengths, such as at 4 or more specific wavelengths, such as at 5 ormore specific wavelengths, such as at 10 or more specific wavelengthsand including detecting the transmitted light at 25 or more specificwavelengths. In some instances, methods include detecting thetransmitted light at one or more of 504 nm, 506 nm, 514 nm, 532 nm, 543nm, 548 nm, 550 nm, 561 nm, 568 nm, 579 nm, 580 nm, 585 nm, 586 nm, 675nm, 710 nm, 808 nm, 815 nm, 830 nm and any combinations thereof. Incertain embodiments, the transmitted light is detected at 548 nm. Inother embodiments, the transmitted light is detected at 675 nm. In yetother embodiments, the transmitted light is detected at 830 nm. In stillother embodiments, the transmitted light is detected at 548 nm and 675nm. In still other embodiments, the transmitted light is detected at 548nm, 675 nm and 830 nm. In still another embodiment, the transmittedlight is detected at 504 nm, 506 nm, 514 nm, 532 nm, 543 nm, 548 nm, 550nm, 561 nm, 568 nm, 579 nm, 580 nm, 585 nm, 586 nm, 650 nm, 675 nm, 710nm, 808 nm, 815 nm and 830 nm.

Depending on the specific assay protocol, transmitted light may bemeasured continuously or in discrete intervals. For example, in someembodiments, measuring transmitted light is continuous throughout theentire time the sample is being assayed. Where measuring the transmittedlight includes measuring two or more wavelengths or wavelength ranges,the wavelengths or wavelength ranges may be all measured simultaneously,or each wavelength or wavelength range may be measured sequentially.

In other embodiments, transmitted light is measured in discreteintervals, such as measuring light transmitted through the sample every0.001 microseconds, every 0.01 microseconds, every 0.1 microseconds,every 1 microsecond, every 10 microseconds, every 100 microseconds andincluding every 1000 microseconds.

Depending on the quality (e.g., homogeneity) of the biological sample,presence of interferents, light source and wavelengths being measured,the light transmitted through sample chamber may be measured one or moretimes during the subject methods, such 2 or more times, such as 3 ormore times, such as 5 or more times and including 10 or more times. Incertain embodiments, the transmitted light is measured two or moretimes, with the data being averaged to calculate absorbance by thetarget analyte, as described below.

In certain embodiments, methods include projecting a slit-shaped beamonto the detector to provide a blank measurement. In these embodiments,the slit-shaped beam may be accomplished by illuminating a blankreference window with the slit-shaped beam and focusing the slit-shapedbeam projected through the blank reference window onto the detector.

For example, the blank reference window may be integrated into thesubject systems (as described below) where light from the light sourceis directed through the integrated blank reference window. In someinstances, light transmitted through the blank reference window isdirected through the objective lens and onto the wavelength separatorand detector. In other instances, light transmitted through the blankreference window is provided directly onto the detector. In someinstances, the blank reference window may be positioned on themicrofluidic cartridge, such as along the same optical axis as thesample chamber.

The absorbance by the blank reference window is in certain embodiments,configured to be identical to absorbance by the sample chamber such thattransmission through the blank reference window can be used to correctfor absorption, scatter, etc. by the microfluidic cartridge whenpracticing the methods described herein. In certain embodiments, theblank reference window has an absorbance and transmission at the one ormore wavelengths of incident light which is substantially the same asthe capillary channel sample chamber. In other embodiments, the blankreference window scatters light at the one or more wavelengths which issubstantially the same as the capillary channel sample chamber. In yetother embodiments, the blank reference window has an absorbance,transmission and scatters light at the one or more incident wavelengthswhich is substantially the same as the capillary channel sample chamber.In still other embodiments, the blank reference window has the sameindex of refraction as the capillary channel sample chamber.

In other embodiments, methods include projecting non-diffracted lightfrom the light source onto the detector to provide for a blank ofincident light. In certain embodiments, the non-diffracted light is usedfor calibrating the detector.

Light transmitted through the sample chamber may be measured by anyconvenient light detecting protocol, including but not limited tophotosensors or photodetectors, such as active-pixel sensors (APSs),avalanche photodiode, image sensors, charge-coupled devices (CCDs),intensified charge-coupled devices (ICCDs), light emitting diodes,photon counters, bolometers, pyroelectric detectors, photoresistors,photovoltaic cells, photodiodes, photomultiplier tubes,phototransistors, quantum dot photoconductors or photodiodes andcombinations thereof, among other photodetectors. In certainembodiments, the transmitted light is measured with a charge-coupleddevice (CCD). Where the transmitted light is measured with a CCD, theactive detecting surface area of the CCD may vary, such as from 0.01 cm²to 10 cm², such as from 0.05 cm² to 9 cm², such as from, such as from0.1 cm² to 8 cm², such as from 0.5 cm² to 7 cm² and including from 1 cm²to 5 cm².

As summarized above, aspects of the present disclosure include methodsfor assaying a sample for one or more analytes. In embodiments, to assayfor the analyte, the absorbance of light by the target analyte iscalculated using the measured transmitted light. In some embodiments,the absorbance is calculated at one or more wavelengths, such as at 2 ormore different wavelengths, such as at 3 or more different wavelengths,such as at 4 or more different wavelengths and including calculating theabsorbance of the target analyte at 5 or more different wavelengths. Forexample, absorbance of the target analyte may be calculated using thedetected transmitted light at one or more of 504 nm, 506 nm, 514 nm, 532nm, 543 nm, 548 nm, 550 nm, 561 nm, 568 nm, 579 nm, 580 nm, 585 nm, 586nm, 675 nm, 710 nm, 808 nm, 815 nm, 830 nm or any combinations thereof.In certain embodiments, absorbance of the analyte is calculated usingthe detected transmitted light at 548 nm. In other embodiments,absorbance of the analyte is calculated using the detected transmittedlight at 675 nm. In yet other embodiments, absorbance of the analyte iscalculated using the detected transmitted light at 830 nm. In stillother embodiments, absorbance of the analyte is calculated using thedetected transmitted light at 548 nm and 675 nm. In still otherembodiments, absorbance of the analyte is calculated using the detectedtransmitted light at 548 nm, 675 nm and 830 nm.

In some embodiments, calculating absorbance of light by the targetanalyte includes calculating absorbance over a range of wavelengths(e.g., 400 nm-800 nm; 495 nm-525 nm; 800 nm-835 nm, etc.). For example,methods may include calculating absorbance over one or more of thewavelength ranges of: 400 nm-800 nm; 498 nm-510 nm; 500 nm-600 nm; 500nm-700 nm; 500 nm-520 nm; 540 nm-550 nm; 545 nm-555 nm; 550 nm-570 nm;550 nm-580 nm; 560 nm-590 nm; 575 nm-595 nm; 580 nm-590 nm; 600 nm-700nm; 600 nm-630 nm; 650 nm-750 nm; 750 nm-850 nm; 810 nm-830 nm; 815nm-825 nm and any combinations thereof. For example, methods includecalculating absorbance of light by the target analyte over thewavelengths ranging from 400 nm-800 nm. In another instance, methodsinclude calculating absorbance of light by the target analyte over thewavelengths ranging from 500 nm-520 nm and 650 nm-750 nm. In anotherinstance, methods include calculating absorbance of light by the targetanalyte over the wavelengths ranging from 540 nm-560 nm and 650 nm-750nm. In yet another instance, methods include calculating absorbance oflight by the target analyte over the wavelengths ranging from 560 nm-590nm and 650 nm-750 nm. In still another instance, methods includecalculating absorbance of light by the target analyte over thewavelengths ranging from 500 nm-520 nm, 560 nm-590 nm, and 650-750 nm.

For example, where the sample is whole blood and the analyte of interestis hemoglobin, the concentration of hemoglobin in whole blood may becalculated by measuring transmitted light at a first and a secondwavelength, where the wavelengths may vary, and include, but are notlimited to, isobestic points, etc. In some instances, the firstwavelength is an isosbestic point for hemoglobin with one or more ofoxyhemoglobin, carboxyhemoglobin, methemoglobin, sulfo-hemoglobin,azide-methemoglobin and cyano-methemoglobin, such as an isosbestic pointfor hemoglobin and oxyhemoglobin or a triple isosbestic point forhemoglobin, oxyhemoglobin and carboxyhemoglobin. For example, a firstwavelength is, in certain instances, 506 nm, 548 nm, 569 nm, 579 nm, 585nm or 586 nm. The second wavelength is, in these embodiments, a lowabsorbing wavelength (e.g., near-IR) and may be also be an isosbesticpoint for hemoglobin with one or more of oxyhemoglobin,carboxyhemoglobin, methemoglobin, sulfo-hemoglobin, azide-methemoglobinand cyano-methemoglobin, such as a isosbestic point for hemoglobin andoxyhemoglobin or a triple isosbestic point for hemoglobin, oxyhemoglobinand carboxyhemoglobin. For example, a second wavelength is, in certaininstances, 650 nm, 675 nm, 710 nm, 785 nm, 808 nm, 815 nm or 830 nm.

The absorbance of light by the target analyte may be calculated usingany convenient priniciple, e.g., the the Beer-Lambert Law:

Absorbance (λ)=−Log₁₀(I/I ₀)

where I is the intensity of the light transmitted through the samplechamber and I₀ is the intensity of incident light used to interrogatethe sample. Depending on the path length of the sample chamber (asdescribed below), the concentration of the analyte can be determinedusing the calculated absorbance of the analyte:

Absorbance (λ)=[molar absorptivity]×[concentration]×[pathlength]

Absorbance may be calculated in conjunction with measurement of thetransmitted light or may be conducted after a predetermined durationfollowing measurement of the transmitted light. In some embodiments,absorbance is continuously calculated in conjunction with measurement ofthe transmitted light, such as where transmitted light is measured atone or more specific wavelengths. Where the subject methods includecalculating absorbance of the analyte at two or more wavelengths, theabsorbance may be calculated at both wavelengths simultaneously orabsorbance may be calculated at each wavelength sequentially.

In other embodiments, absorbance is calculated a predetermined durationafter measurement of the transmitted light, such as 0.001 seconds orlonger after measurement, such as 0.01 seconds or longer aftermeasurement, such as 0.1 seconds or longer after measurement, such as0.5 seconds or longer after measurement, such as 1 second or longerafter measurement and including 5 seconds or longer after measurement ofthe transmitted light. For example, in embodiments where a spectrum overa range of wavelengths is collected, absorbance may be calculated apredetermined duration after the entire spectrum is collected.

In some embodiments, methods include calculating concentration based onabsorbance determined from the transmitted light. In embodiments, theconcentration of the analyte can be calculated using the absorbance atany desired wavelength, such as at two or more wavelengths, such as atthree or more wavelengths and including at five or more wavelengths. Insome embodiments, concentration of the analyte in the sample iscalculated at one or more peak absorbance values in the absorbancespectrum. In other embodiments, concentration of the analyte in thesample is calculated using one or more wavelengths where the analyte hasthe largest molar absorptivity. Where two or more analytes are ofinterest, the concentration of the analytes may, in certain instances,be calculated using the absorbance at a wavelength corresponding to anisosbestic point for the two or more analytes.

In calculating concentration of the analyte, the absorbance of light bythe target analyte is first determined using the Beer-Lambert Law:

Absorbance (λ)=−Log₁₀(I/I ₀)

where I is the intensity of the light transmitted through the samplechamber and I₀ is the intensity of incident light used to interrogatethe sample. Depending on the path length of the sample chamber, theconcentration of the analyte is then determined using the calculatedabsorbance of the analyte:

Absorbance (λ)=[molar absorptivity]×[concentration]×[pathlength]

In one embodiment, methods include calculating concentration of theanalyte while accounting for scatter by the sample by measuringtransmitted light at a first and a second wavelength. The firstwavelength, in some instances, is a wavelength where the analyte has ahigh molar absorptivity. In other instances, the first wavelength is awavelength at an isosbestic point with one or more derivatives of theanalyte that are included in the analyte concentration. The secondwavelength is, in these embodiments, a wavelength where the analyte haslow molar absorptivity. The second wavelength may also be a wavelengthat an isosbestic point with one or more derivatives of the analyte. Tocalculate concentration of the analyte accounting for scatter:

Concentration_(analyte) =A*(Abs_(λ1))+B*(Abs_(λ2))+C,

where A, B, and C are coefficients which depend on the wavelengthsinterrogated and analytes being measured. In embodiments, the value of Amay vary, in certain instances, ranging from 20 g/dL to 60 g/dL, such asfrom 25 g/dL to 57.5 g/dL, such as from 30 g/dL to 55 g/dL, such as from35 g/dL to 50 g/dL and including from 37.5 g/dL to 45 g/dL. The value ofB may also vary, in certain instances, ranging from 0.01 g/dL to 5 g/dL,such as from 0.05 g/dL to 4.5 g/dL, such as from 0.1 g/dL to 4 g/dL,such as from 0.25 g/dL to 3.5 g/dL, such as from 0.5 g/dL to 3 g/dL andincluding from 0.5 g/dL to 2 g/dL. Likewise, the value of C may alsovary, ranging from 0.01 g/dL to 2 g/dL, such as from 0.025 g/dL to 1.75g/dL, such as from 0.05 g/dL to 1.5 g/dL, such as from 0.1 g/dL to 1.25g/dL and including from 0.25 g/dL to 2 g/dL.

As discussed above, in certain embodiments the sample is whole blood andthe analyte of interest is hemoglobin. In some instances, methodsinclude calculating concentration of total hemoglobin in whole bloodwhile accounting for scatter by the sample by measuring transmittedlight at a first and a second wavelength. The first wavelength may be awavelength where hemoglobin has a high molar absorptivity. In someinstances, the first wavelength may be an isosbestic point forhemoglobin with one or more of oxyhemoglobin, carboxyhemoglobin,methemoglobin, sulfo-hemoglobin, azide-methemoglobin andcyano-methemoglobin, such as a isosbestic point for hemoglobin andoxyhemoglobin or a triple isosbestic point for hemoglobin, oxyhemoglobinand carboxyhemoglobin. For example, a first wavelength is, in certaininstances, 506 nm, 548 nm, 569 nm, 579 nm, 585 nm or 586 nm. To accountfor scatter, a second wavelength where hemoglobin has low absorptivitymay be chosen, such as a near infrared wavelength. In some instances,the second wavelength is an isosbestic point for hemoglobin with one ormore of oxyhemoglobin, carboxyhemoglobin, methemoglobin,sulfo-hemoglobin, azide-methemoglobin and cyano-methemoglobin, such as aisosbestic point for hemoglobin and oxyhemoglobin or a triple isosbesticpoint for hemoglobin, oxyhemoglobin and carboxyhemoglobin. For example,a second wavelength is, in certain instances, 650 nm, 675 nm, 710 nm,785 nm, 808 nm, 815 nm or 830 nm.

For example, where the first wavelength is 548 nm and the secondwavelength is 675 nm, to calculate concentration of hemoglobinaccounting for scatter:

Concentration_(Hb) =A*(Abs_(548 nm))+B*(Abs_(675 nm))+C,

In embodiments, the value of A for a whole blood sample may vary, incertain instances, ranging from 20 g/dL to 60 g/dL, such as from 25 g/dLto 57.5 g/dL, such as from 30 g/dL to 55 g/dL, such as from 35 g/dL to50 g/dL and including from 37.5 g/dL to 45 g/dL. The value of B for awhole blood sample may also vary, in certain instances, ranging from0.01 g/dL to 5 g/dL, such as from 0.05 g/dL to 4.5 g/dL, such as from0.1 g/dL to 4 g/dL, such as from 0.25 g/dL to 3.5 g/dL, such as from 0.5g/dL to 3 g/dL and including from 0.5 g/dL to 2 g/dL. Likewise, thevalue of C of a whole blood sample may also vary, ranging from 0.01 g/dLto 2 g/dL, such as from 0.025 g/dL to 1.75 g/dL, such as from 0.05 g/dLto 1.5 g/dL, such as from 0.1 g/dL to 1.25 g/dL and including from 0.25g/dL to 2 g/dL.

In another example, where the first wavelength is 548 nm and the secondwavelength is 650 nm, to calculate concentration of hemoglobinaccounting for scatter:

Concentration_(Hb) =A*(Abs_(548 nm))+B*(Abs_(650 nm))+C,

In embodiments, the value of A for a whole blood sample may vary, incertain instances, ranging from 20 g/dL to 60 g/dL, such as from 25 g/dLto 57.5 g/dL, such as from 30 g/dL to 55 g/dL, such as from 35 g/dL to50 g/dL and including from 37.5 g/dL to 45 g/dL. The value of B for awhole blood sample may also vary, in certain instances, ranging from0.01 g/dL to 5 g/dL, such as from 0.05 g/dL to 4.5 g/dL, such as from0.1 g/dL to 4 g/dL, such as from 0.25 g/dL to 3.5 g/dL, such as from 0.5g/dL to 3 g/dL and including from 0.5 g/dL to 2 g/dL. Likewise, thevalue of C of a whole blood sample may also vary, ranging from 0.01 g/dLto 2 g/dL, such as from 0.025 g/dL to 1.75 g/dL, such as from 0.05 g/dLto 1.5 g/dL, such as from 0.1 g/dL to 1.25 g/dL and including from 0.25g/dL to 2 g/dL.

In another embodiment, methods include calculating concentration of theanalyte while accounting for interferents by measuring the transmittedlight and determining the absorbance at a first wavelength and a secondwavelength. In this embodiment, a concentration of the analyte iscalculated by determining the concentration from the absorbance at thefirst wavelength and determining the concentration from the absorbanceat the second wavelength and subtracting the concentration obtained atsecond wavelength from the concentration at the first wavelength.

In certain embodiments, the subject methods may be coupled with methodsfor interrogating the sample for one or more analytes by a fluorescenceassay. For example, as described in greater detail below, the sample maybe contacted with one or more reagents having fluorescent markers, theemission being detectable by one or more photosensors or photodetectors.As such, aspects of the present disclosure according to certainembodiments include assaying a sample for one or more analytes bycontacting the sample with one or more reagents and opticallyinterrogating the sample by an absorption assay (as discussed above) incombination with a fluorescence assay.

In embodiments, samples assayed for fluorescence may be illuminated withone or more light sources. Depending on the target analyte, the samplemay be illuminated with one or more broadband light sources (e.g., ahalogen lamp, deuterium arc lamp, xenon arc lamp, stabilizedfiber-coupled broadband light source, a broadband LED with continuousspectrum, superluminescent emitting diode, semiconductor light emittingdiode, wide spectrum LED white light source, an multi-LED integratedwhite light source, combinations thereof, as described above) or may beilluminated with one or more narrow band light sources emitting aparticular wavelength or narrow range of wavelengths (e.g., narrowwavelength LED, laser diode or a broadband light source coupled to oneor more optical bandpass filters, diffraction gratings, monochromators,combination thereof, as described above).

Depending on the dimensions and positioning of the sample chamber, theangle of incident illumination for fluorescence assay may vary, rangingfrom 30° to 60° with respect to the plane of the sample chamber, such asfrom 35° to 55°, such as from 40° to 50° and including illuminating thesample chamber at a 45° with respect to the plane of the sample chamber.

Where more than one light source is employed, the sample may beilluminated with the light sources simultaneously or sequentially, or acombination thereof. Where the sample is sequentially illuminated withtwo or more light sources, the time each light source illuminates thesame may independently be 0.001 seconds or more, such as 0.01 seconds ormore, such as 0.1 seconds or more, such as 1 second or more, such as 5seconds or more, such as 10 seconds or more, such as 30 seconds or moreand including 60 seconds or more. In embodiments where the sample issequentially illuminated by two or more light sources, the duration thesample is illuminated by each light source may be the same or different.The time period between illumination by each light source may also vary,as desired, being separated independently by a delay of 1 second ormore, such as 5 seconds or more, such as by 10 seconds or more, such asby 15 seconds or more, such as by 30 seconds or more and including by 60seconds or more.

Depending on the specific analytes being assayed as well as the reagentsand fluorescent markers employed, illumination of the sample may becontinuous or in discrete intervals. For example, in some embodiments,the sample may be illuminated continuously throughout the entire timethe sample is being assayed. In other embodiments, the sample may beilluminated in regular intervals, such as illuminating the sample every0.001 microseconds, every 0.01 microseconds, every 0.1 microseconds,every 1 microsecond, every 10 microseconds, every 100 microseconds andincluding every 1000 microseconds. The sample may be illuminated withthe light source one or more times at any given measurement period, suchas 2 or more times, such as 3 or more times, including 5 or more timesat each measurement period.

During fluorescence assay, the sample may be illuminated with abroadband light source having wavelengths ranging from 300 nm to 900 nm,such as from 325 nm to 875 nm, such as from 350 nm to 850 nm, such asfrom 375 nm to 825 nm and including from 400 nm to 800 nm or some otherrange. In other embodiments, the sample is illuminated with specificwavelengths of light or a narrow range of specific wavelengths (such aswith a narrow band lamp or LED). For example, the sample may beilluminated for fluorescence with a narrow band light source or one ormore monochromatic LEDs emitting light in the range of 450 nm to 700 nm,such as at 480 nm, 565 nm and 650 nm.

As the subject fluorescence assay is coupled with methods for absorptionassay described above, emitted light from the sample may be collected,spatially separated into component wavelengths and detected in a similarmanner as described above. As described in detail below, fluorescencedetection systems and the absorbance detection systems employ one ormore common components as described herein. For example, in someinstances both fluorescence assay and absorbance assay employ a commonobjective lens module for collecting and focusing light from the samplechamber (e.g., emitted light or transmitted light). In other instances,both fluorescence assay and absorbance assay employ a common wavelengthseparation protocol (e.g., diffraction grating, optical filters, filterwheel having one or more diffraction gratings and optical filters) tospatially separate the collected light into component wavelengths. Inyet other instances, both fluorescence assay and absorbance assay employthe same detection protocol for measuring light (e.g., emitted light ortransmitted light) from the sample chamber.

In certain embodiments, the subject fluorescence assay may includemethods for imaging samples in capillary channels such as thosedescribed in U.S. Pat. Nos. 8,248,597; 7,927,561 and 7,738,094 as wellas those described in co-pending U.S. patent application Ser. No.13/590,114 filed Aug. 20, 2012, the disclosures of which are hereinincorporated by reference.

In certain specific embodiments, the subject methods provide anabsorbance assay for hemoglobin. As discussed above, hemoglobin may bepresent in any type of diagnostic sample, such as supernatants, lysates,buffered solution, as well as in biological samples including wholeblood. In accordance with the methods described above, an amount of thesample is loaded into a sample chamber and illuminated through a slitprojection module with one or more light sources, with light transmittedthrough the whole blood sample in the sample chamber being collected andspatially separated into component wavelengths for detection. Dependingon the size of the whole blood sample, the sample chamber may be amicrofluidic capillary channel sample chamber. Hemoglobin absorbance canbe determined from the transmitted light at one or more wavelengths oralternatively, an entire spectrum of hemoglobin absorption may becalculated. Based on the absorbance at one or more wavelengths, thehemoglobin concentration in the whole blood sample can be determined inthese embodiments of the subject methods.

In certain other specific embodiments, the subject methods provide areagent free hemoglobin absorbance assay. By “reagent free” is meantthat the assay of hemoglobin employs no reagents that interact or areused to visualize hemoglobin in the sample. As such, hemoglobin(including derivatives such oxyhemoglobin and carboxyhemoglobin) isassayed in its native state without reagent modification. In theseinstances, an unaltered whole blood sample is loaded into a samplechamber and illuminated with one or more light sources through a slitprojection module, with light transmitted through the whole blood samplein the sample chamber being collected and spatially separated intocomponent wavelengths for detection. Depending on the size of the wholeblood sample, the sample chamber may be a microfluidic capillary channelsample chamber. Hemoglobin absorbance can be detected at one or morewavelengths or alternatively, an entire spectrum of hemoglobinabsorption may be calculated. Based on the absorbance at one or morewavelengths, the hemoglobin concentration in the unaltered whole bloodsample can be determined in these embodiments of the subject methods.

In certain other specific embodiments, the subject methods provide ahemoglobin absorbance assay on a sample also being assayed for one ormore additional analytes, such as for example cell surface markers. Inthese embodiments, one or more reagents, including specific bindingmembers, enzymes, substrates, oxidizers as well as binding moleculescoupled to one or more fluorescent markers are contacted with the wholeblood and the reagent-mixed whole blood sample is loaded into a samplechamber. The loaded sample chamber (such as a microfluidic capillarychannel sample chamber) is illuminated with one or more light sourcesthrough a slit projection module, with light transmitted through thewhole blood sample in the sample chamber being collected and spatiallyseparated into component wavelengths for detection. Hemoglobinabsorbance can be detected at one or more wavelengths or alternatively,an entire spectrum of hemoglobin absorption may be calculated. Based onthe absorbance at one or more wavelengths, the hemoglobin concentrationin the reagent-mixed whole blood sample can be determined in theseembodiments of the subject methods. In conjunction with assaying forhemoglobin in the reagent-mixed sample, one or more additional analytesmay be assayed. In some instances, the subject methods provide afluorescence assay performed in conjunction with the hemoglobinabsorbance assay to assay for one or more cell surface markers bindingto the one or more reagents mixed into the whole blood sample. In theseinstances, a fluorescence light source illuminates the sample chamberloaded with reagent-mixed whole blood sample and fluorescence emissionfrom fluorescence tags bound to target analytes is collected andspatially separated for detection.

In certain other specific embodiments, the subject methods provide ahemoglobin absorbance assay on a sample for which is also fluorescenceassayed for CD4 and % CD4. In these instances, the whole blood sample isapplied to the sample application site of a microfluidic cartridgehaving a capillary channel sample chamber. The applied sample is carriedthrough the inlet of the microfluidic capillary channel into a reagentmixing chamber having a porous disc for contacting the reagent mixturewith the whole blood sample. The reagent mixture, in these instances,includes dried storage stable reagents CD4-PECy5, CD3-APC, CD45RA-APCand CD14-PE. The reagent mixed whole blood sample is carried bycapillary action through to the sample chamber where the sample chamberis illuminated for hemoglobin assay by two light sources, a broadbandwhite light LED and a near-infrared LED through a slit projection modulewhich is moved laterally across the sample chamber. Light transmittedthough the sample chamber is collected with an objective, magnifyinglens and autofocused onto a diffraction grating to spatially separatethe transmitted light on the surface of a CCD detector. The absorbanceat two wavelengths, 548 nm and 675 nm are determined and the totalhemoglobin absorbance accounting for scatter is calculated to assay forhemoglobin.

The reagent mixed whole blood sample in the capillary channel samplechamber is also assayed for CD4 by detecting fluorescence by fluorescenttags in the reagent mixture. CD4 may be assayed for by illuminating thereagent mixed whole blood sample in the capillary channel sample chamberwith a light source and emission from the fluorescent tags in thereagent mixed whole blood sample is collected with a common objective,magnifying lens and autofocused onto the surface of the CDD detector.CD4 cell counting is then conducted by fluorescent image cytometry.

In certain embodiments, aspects of the methods include applying a sampleto a microfluidic device configured to perform an assay of a liquidsample having a sample application site, an inlet for inputting thesample from the sample application site, a reagent contacting chamberfor contacting the sample with one or more reagents and a capillarysample chamber in fluid communication with the reagent contactingchamber.

In some embodiments, the sample is applied to the application site ofthe microfluidic device and allowed to flow through the inlet and intothe reagent contacting chamber, followed by flow through the capillarysample chamber such that sufficient sample is provided to beinterrogated in the capillary sample chamber as described above.

In certain instances, methods include providing a sample contactedsample application site of the microfluidic device. By “sample-contactedsample application site” is meant a sample application site that hasbeen contacted by the sample. In practicing methods of the presentdisclosure, a sample-contacted application site is provided by applyinga sample to the sample application site of the microfluidic device. Theamount of sample that is applied to the sample application site mayvary, so long as it is sufficient to provide desired capillary flowthrough the sample chamber and adequate sample for interrogation by thesubject methods described herein. For example, the amount of sampleapplied to the application site may range from 0.01 μl_(—) to 1000 μL,such as from 0.05 μl_(—) to 900 μL, such as from 0.1 μl_(—) to 800 μL,such as from 0.5 μl_(—) to 700 μL, such as from 1 μl_(—) to 600 μL, suchas from 2.5 μl_(—) to 500 μL, such as from 5 μl_(—) to 400 μL, such asfrom 7.5 μl_(—) to 300 μl_(—) and including from 10 μl_(—) to 200 μl_(—)of sample.

The sample may be applied to the sample application site using anyconvenient protocol, e.g., via dropper, pipette, syringe and the like.The sample may be applied in conjunction or incorporated into a quantityof a suitable liquid, e.g., buffer, to provide for adequate fluid flow.Any suitable liquid may be employed, including but not limited tobuffers, cell culture media (e.g., DMEM), etc. Buffers include, but arenot limited to: tris, tricine, MOPS, HEPES, PIPES, MES, PBS, TBS, andthe like. Where desired, detergents may be present in the liquid, e.g.,NP-40, TWEEN™ or TritonX100 detergents.

In certain embodiments, the sample-contacted sample application site isprovided by combining the sample with one or more assay components(e.g., a reagent, a buffer, and the like) prior to applying the samplewhich has the assay component(s) to the sample application site. Whenthe sample is combined with one or more assay components prior to theapplication of the sample having the assay component(s) to the sampleapplication site, the combination may be achieved using any convenientprotocol. The amount of an assay component(s), when combined with thesample, may vary as desired. In some embodiments, the sample-contactedsample application site is provided by applying one or more assaycomponents to the sample receiving application site prior to applyingthe sample to the sample application site. In some embodiments, thesample-contacted sample application site is provided by applying thesample to the sample application site prior to applying one or moreassay components to the sample application site. As mentioned above, insome embodiments, the device includes one or more assay components(e.g., reagent). In such cases, the sample-contacted sample applicationsite is provided by applying the sample to the sample application site,e.g., without prior combination with one or more assay components.

Following sample application, the sample is allowed to flow through thecapillary sample chamber, and one or more portions of the channel, e.g.,the detection region, including the entire channel, is then interrogatedassay for the target analyte(s) in the sample. Depending on the targetanalyte and presence of one or more reagents, the sample may beinterrogated immediately after sample application or following apredetermined period of time after sample application, such as a periodof time ranging from 10 seconds to 1 hour, such as 30 seconds to 30minutes, e.g., 30 seconds to 10 minutes, including 30 seconds to 1minute.

One example of suitable methods and microfluidic devices for preparing asample for interrogation by the subject methods may include, but are notlimited to those described in copending U.S. patent application Ser. No.14/152,954 filed on Jan. 10, 2014, the disclosure of which is hereinincorporated by reference.

Systems for Assaying a Sample for an Analyte

Aspects of the present disclosure further include systems for practicingthe subject methods. In embodiments, systems which include a lightsource, a slit projection module (e.g., a slit for narrowing a beam oflight or a slit coupled to a focusing lens that focuses the narrowedlight) and a detector for detecting one or more wavelengths of thetransmitted light are provided. In certain embodiments, systems furtherinclude a microfluidic device for preparing and providing the sample forassay using the subject systems.

As summarized above, aspects of the present disclosure include assayinga sample for one or more analytes. Systems include one or more lightsources for interrogating a sample chamber containing a sample ofinterest. In some embodiments, the light source is a broadband lightsource, emitting light having a broad range of wavelengths, such as forexample, spanning 50 nm or more, such as 100 nm or more, such as 150 nmor more, such as 200 nm or more, such as 250 nm or more, such as 300 nmor more, such as 350 nm or more, such as 400 nm or more and includingspanning 500 nm or more. For example, one suitable broadband lightsource emits light having wavelengths from 400 nm to 800 nm. Anotherexample of a suitable broadband light source includes a light sourcethat emits light having wavelengths from 500 nm to 700 nm. Anyconvenient broadband light source protocol may be employed, such as ahalogen lamp, deuterium arc lamp, xenon arc lamp, stabilizedfiber-coupled broadband light source, a broadband LED with continuousspectrum, superluminescent emitting diode, semiconductor light emittingdiode, wide spectrum LED white light source, an multi-LED integratedwhite light source, among other broadband light sources or anycombination thereof.

In other embodiments, the light source is a narrow band light sourceemitting a particular wavelength or a narrow range of wavelengths. Insome instances, the narrow band light sources emit light having a narrowrange of wavelengths, such as for example, 50 nm or less, such as 40 nmor less, such as 30 nm or less, such as 25 nm or less, such as 20 nm orless, such as 15 nm or less, such as 10 nm or less, such as 5 nm orless, such as 2 nm or less and including light sources which emit aspecific wavelength of light (i.e., monochromatic light). Any convenientnarrow band light source protocol may be employed, such as a narrowwavelength LED, laser diode or a broadband light source coupled to oneor more optical bandpass filters, diffraction gratings, monochromatorsor any combination thereof.

The subject systems may include one or more light sources, as desired,such as two or more light sources, such as three or more light sources,such as four or more light sources, such as five or more light sourcesand including ten or more light sources. The light source may include ancombination of types of light sources, for example, where two lightssources are employed, a first light source may be a broadband whitelight source (e.g., broadband white light LED) and second light sourcemay be a broadband near-infrared light source (e.g., broadband near-IRLED). In other instances, where two light sources are employed, a firstlight source may be a broadband white light source (e.g., broadbandwhite light LED) and the second light source may be a narrow spectralight source (e.g., a narrow band visible light or near-IR LED). In yetother instances, the light source is an plurality of narrow band lightsources each emitting specific wavelengths, such as an array of two ormore LEDs, such as an array of three or more LEDs, such as an array offive or more LEDs, including an array of ten or more LEDs.

In some embodiments, light sources emit light having wavelengths rangingfrom 400 nm to 900 nm, such as from 450 nm to 850 nm, such as from 500nm to 800 nm, such as from 550 nm to 750 nm and including from 600 nm to700 nm. For example, the light source may include a broadband lightsource emitting light having wavelengths from 400 nm to 900 nm. In otherinstances, the light source includes a plurality of narrow band lightsources emitting wavelengths ranging from 400 nm to 900 nm. For example,the light source may be plurality of narrow band LEDs (1 nm-25 nm) eachindependently emitting light having a range of wavelengths between 400nm to 900 nm.

In certain embodiments, systems include two broadband light sources,configured to collectively emit light having wavelengths ranging from400 nm to 900 nm. For example, the light sources may be a white lightLED emitting light having wavelengths ranging from 400 nm to 700 nm anda near-infrared LED emitting light having wavelengths ranging from 700nm to 900 nm. In some embodiments, the irradiation profile of each lightsource may vary, having any number of emission peaks. In certaininstances, the light source includes a white light LED emitting lighthaving wavelengths ranging from 400 nm to 700 nm and having emissionpeaks at about 450 nm and 550 nm and a near-infrared LED emitting lighthaving wavelengths ranging from 700 nm to 900 nm and having an emissionpeak at about 830 nm.

In other embodiments, the light source is a plurality of narrow bandlamps or LEDs each independently emitting specific wavelengths of lightin the range of 400 nm to 900 nm. In one example, the narrow band lightsource is one or more monochromatic LEDs emitting light in the range of500 nm to 700 nm, such as at 504 nm, 506 nm, 514 nm, 532 nm, 543 nm, 548nm, 550 nm, 561 nm, 568 nm, 579 nm, 580 nm, 585 nm, 586 nm or anycombination thereof. In another example, the narrow band light source isan array of LEDs emitting light with wavelengths ranging from 400 nm to900 nm. In another example, the narrow band light source is one or morenarrow band lamps emitting light in the range of 500 nm to 700 nm, suchas a narrow band cadmium lamp, cesium lamp, helium lamp, mercury lamp,mercury-cadmium lamp, potassium lamp, sodium lamp, neon lamp, zinc lampor any combination thereof.

As summarized above, systems include a slit projection module configuredto narrow a beam of light and produce a beam of light in the shape of aslit projected onto the sample chamber. In some embodiments, the slitprojection module includes a slit. In other embodiments, the slitprojection module includes a slit coupled to a focusing lens configuredto focus the narrow slit-shaped beam of light at the sample chamber.

In some embodiments, systems of the present disclosure are configuredsuch that the sample chamber, the slit projection module or both thesample chamber and slit projection module may be moved to displace theslit-shaped beam of light across the sample chamber. Where movement ofthe slit-shaped beam of light across the sample chamber is desired, insome embodiments systems are configured to move the sample chamber whilethe slit-projection module is maintained in a stationary position. Inother embodiments, systems are configured to move the slit-projectionand the sample chamber is maintained in a stationary. In yet otherembodiments, the system is configured to move both the slit projectionmodule and the sample chamber. Any displacement protocol may be employedin the subject systems to move the slit-shaped beam of light across thesample chamber, such as manually (i.e., movement of the sample chamberor slit projection module directly by hand), with assistance by amechanical device or by a motor actuated displacement device. Forexample, in some embodiments the sample chamber is moved in the subjectsystems with a mechanically actuated translation stage, mechanicalleadscrew assembly, mechanical slide device, mechanical lateral motiondevice, mechanically operated geared translation device. In otherembodiments, the sample chamber is moved with a motor actuatedtranslation stage, leadscrew translation assembly, geared translationdevice, such as those employing a stepper motor, servo motor, brushlesselectric motor, brushed DC motor, micro-step drive motor, highresolution stepper motor, among other types of motors. In certaininstances, the sample chamber is housed in a cartridge holder which isoperably or mechanically connected to the translation or displacementdevice. In these instances, the sample chamber (or microcartridgecontaining the sample chamber) is first loaded into the cartridgehousing and the entire housing is displaced during the subject methods.

Likewise, the slit projection module, in certain instances, is moved inthe subject systems with a mechanically actuated translation stage,mechanical leadscrew assembly, mechanical slide device, mechanicallateral motion device, mechanically operated geared translation device.In other embodiments, the slit projection module is moved with amotor-actuated translation stage, leadscrew translation assembly, gearedtranslation device, such as those employing a stepper motor, servomotor, brushless electric motor, brushed DC motor, micro-step drivemotor, high resolution stepper motor, among other types of motors.

In some embodiments, the system is configured to move the sample chamberrelative to a stationary slit projection module to displace theslit-shaped beam laterally across the sample chamber in a singledirection during interrogation of the sample. In other embodiments, thesystem is configured to move the sample chamber relative to a stationaryslit projection module to displace the slit-shaped beam laterally acrossthe sample chamber in back-and-forth motion during interrogation of thesample. For example, the sample chamber may be moved such that theslit-shaped beam is moved along 50% or more of the sample chamber, suchas 55% or more, such as 60% or more, such as 65% or more, such as 70% ormore, such as 75% or more, such as 80% or more, such as 85% or more,such as 90% or more, such as 95% or more, such as 97% or more andincluding along 99% or more of the length of the sample chamber. Incertain instances, the sample chamber is moved such that slit-shapedbeam is moved along substantially the entire length of the samplechamber.

In other embodiments, the system is configured to move the slitprojection module relative to a stationary sample chamber to displacethe slit-shaped beam laterally across the sample chamber in a singledirection during interrogation of the sample. In other embodiments, thesystem is configured to move the slit projection module relative to astationary sample chamber to displace the slit-shaped beam laterallyacross the sample chamber in back-and-forth motion during interrogationof the sample. For example, the slit projection module may be moved suchthat the slit-shaped beam is moved along 50% or more of the samplechamber, such as 55% or more, such as 60% or more, such as 65% or more,such as 70% or more, such as 75% or more, such as 80% or more, such as85% or more, such as 90% or more, such as 95% or more, such as 97% ormore and including along 99% or more of the length of the samplechamber. In certain instances, the slit projection module is moved suchthat slit-shaped beam is moved along substantially the entire length ofthe sample chamber. In yet other embodiments, the system is configuredto move both the slit projection module and the sample chamber todisplace the slit-shaped beam laterally across the sample chamber in asingle direction during interrogation of the sample. In otherembodiments, the system is configured to move both the slit projectionmodule and sample chamber to displace the slit-shaped beam laterallyacross the sample chamber in back-and-forth motion during interrogationof the sample. For example, the slit projection module and the samplechamber may be both moved such that the slit-shaped beam is moved along50% or more of the sample chamber, such as 55% or more, such as 60% ormore, such as 65% or more, such as 70% or more, such as 75% or more,such as 80% or more, such as 85% or more, such as 90% or more, such as95% or more, such as 97% or more and including along 99% or more of thelength of the sample chamber. In certain instances, the slit projectionmodule and sample chamber are both moved such that slit-shaped beam ismoved along substantially the entire length of the sample chamber.

The slit aperture may be any convenient shape, including but not limitedto an oval, rectangle or other suitable polygon. In certain embodiments,the slit aperture is rectangular. Depending on the size of the samplechamber and slit-shaped beam provided by the light source as well as thedistance between the slit projection module, light source, samplechamber and detector, the dimensions of the slit aperture may vary,having a length which ranges from 1 mm to 10 mm, such as from 1.25 mm to9.5 mm, such as from 1.5 mm to 9 mm, such as from 2 mm to 8 mm, such asfrom 2.5 mm to 7 mm, such as from 3 mm to 6 mm and including from 3.5 mmto 5 mm. The width of the slit aperture may range from 1 μm to 250 μm,such as from 2 μm to 225 μm, such as from 5 μm to 200 μm, such as from10 μm to 150 μm, and including from 15 μm to 125 μm, for example a slithaving an aperture width of 100 μm. Any convenient slit device may beemployed so long as it is sufficient to provide the desired slit-shapedbeam of light for interrogating the sample chamber. For example, theslit may be gold, silver, gold-plated copper, ceramic, chromium, copper,molybdenum and tungsten.

In some instances, the slit projection module also includes an opticaladjustment protocol. By “optical adjustment” is meant that the beam oflight in the shape of a slit may be changed as desired, such as toincrease or decrease the dimensions or to enhance the optical resolutionof the slit shaped beam. In some instances, optical adjustment is amagnification protocol configured to increase the width of the slit,such as by 5% or greater, such as by 10% or greater, such as by 25% orgreater, such as by 50% or greater and including increasing the width ofthe slit shaped beam by 75% or greater. In other instances, opticaladjustment is a de-magnification protocol configured to decrease thewidth of the slit, such as by 5% or greater, such as by 10% or greater,such as by 25% or greater, such as by 50% or greater and includingdecreasing the width of the slit shaped beam by 75% or greater. Incertain embodiments, optical adjustment is an enhanced resolutionprotocol configured to improve the resolution of the slit shaped beam,such as by 5% or greater, such as by 10% or greater, such as by 25% orgreater, such as by 50% or greater and including enhancing theresolution of the slit shaped beam by 75% or greater. The slit shapedbeam may be adjusted with any convenient optical adjustment protocol,including but not limited to lens, mirrors, pinholes, slits, andcombinations thereof.

In certain embodiments, the slit projection module may also include afocusing lens coupled to the slit that is configured to focus thenarrowed slit-shaped beam of light. In some embodiments, the focusinglens is a de-magnifying lens having a magnification ratio ranging from0.1 to 0.95, such as a magnification ratio of from 0.2 to 0.9, such as amagnification ratio of from 0.3 to 0.85, such as a magnification ratioof from 0.35 to 0.8, such as a magnification ratio of from 0.5 to 0.75and including a magnification ratio of from 0.55 to 0.7, for example amagnification ratio of 0.6. For example, the focusing lens is, incertain instances, a double achromatic de-magnifying lens having amagnification ratio of about 0.6. Depending on the distance between theslit projection module, light source, sample chamber and detector aswell as the size of the sample chamber and desired size of slit-shapedbeam, the focal length of the focusing lens may vary, ranging from 5 mmto 20 mm, such as from 6 mm to 19 mm, such as from 7 mm to 18 mm, suchas from 8 mm to 17 mm, such as from 9 mm to 16 and including a focallength ranging from 10 mm to 15 mm. In certain embodiments, the focusinglens has a focal length of about 13 mm.

In some embodiments, the slit and the focusing lens are in opticalcommunication, but are not physically in contact. Depending on the sizeof the sample chamber as well as the desired shape and size of theslit-shaped beam projected onto the sample chamber, the slit may bepositioned a distance from the focusing lens which varies and may be 0.1mm or more, such as 0.2 mm or more, such as 0.5 mm or more, such as 1 mmor more, such as 5 mm or more, such as 10 mm or more, such as 25 mm ormore, such as 50 mm or more, including 100 mm or more. In otherembodiments, the slit is physically coupled to the focusing lens, suchas with an adhesive, co-molded together or integrated together in ahousing having the focusing lens positioned adjacent to the slit. Assuch, the slit and focusing lens may be integrated into a single unit.

As described above, the slit projection module is configured to providea slit-shaped beam having a length and width which varies. In someembodiments, the slit projection module is configured to provide aslit-shaped beam having a length which ranges from 1 mm to 5 mm, such asfrom 1.5 mm to 4.5 mm, such as from 2 mm to 4 mm, such as from 2.5 mm to3.5 mm and including a slit-shaped beam having a length of 3 mm. Inthese embodiments, the slit projection module is configured to provide aslit-shaped beam having a width which ranges from 10 μm to 100 μm, suchas from 15 μm to 95 μm, such as from 20 μm to 90 μm, such as from 25 μmto 85 μm, such as from 30 μm to 80 μm, such as from 35 μm to 75 μm, suchas from 40 μm to 70 μm, such as from 45 μm to 65 μm, and including from50 μm to 60 μm.

As described above, in some embodiments the slit projection module isconfigured to provide a slit-shaped beam having a length that isorthogonal to the length of the sample chamber. Depending on the size ofthe sample chamber, as described below, the slit-projection module maybe configured to provide a slit-shaped beam having a length that is 50%or more of the width of the sample chamber, such as 55% or more, such as60% or more, such as 65% or more, such as 70% or more, such as 75% ormore, such as 80% or more, such as 85% or more, such as 90% or more,such as 95% or more, such as 97% or more and including a slit projectionmodule configured to provide a slit-shaped beam having a length that is99% or more of the width of the sample chamber. In certain instances,the slit projection module is configured to provide a slit-shaped beamthat has a length which is substantially the same as width of the samplechamber. In other embodiments, the slit-projection module is configuredto provide a slit-shaped beam projection which has a length that isgreater than the width of the sample chamber. For example, the slitprojection module is, in certain instances, configured to provide aslit-shaped beam of light that has a length which is 1% or greater thanthe width of the sample chamber, such as 2% or greater, such as 5% orgreater, such as 10% or greater, such as 15% or greater, such as 20% orgreater and including a length which is 25% greater than the width ofthe sample chamber. In yet other instances, the slit projection moduleis configured to provide a slit-shaped beam of light that has a lengthwhich is less than the width of the sample chamber, such as a lengththat is 1% or greater less than the width of the sample chamber, such asa length that is 2% or greater less than the width of the samplechamber, such as a length that is 5% or greater less than the width ofthe sample chamber, such as a length that is 10% or greater less thanthe width of the sample chamber, such as a length that is 15% or greaterless than the width of the sample chamber, such as a length that is 20%or greater less than the width of the sample chamber and including alength that 25% or greater less than the width of the sample chamber.

As discussed in greater detail below, in certain embodiments the subjectsystems are configured to receive a microfluidic cartridge device havinga capillary sample chamber. In these embodiments, systems may alsoinclude a cartridge holder for receiving the cartridge into the systemFor example, the cartridge holder may include a support for receivingthe microfluidic cartridge device and one or more cartridge retainersfor maintaining the microfluidic cartridge device in the cartridgeholder. In some instances, the cartridge holder includes vibrationdampers for reducing agitation of the microfluidic cartridge devicepositioned in the cartridge holder as well as one or more cartridgepresence flags configured to indicate that a microfluidic cartridgedevice is present in the cartridge holder.

Where the subject systems are configured to move the sample chamberduring interrogation (as discussed above), systems may also include acartridge shuttle coupled to the cartridge holder for moving themicrofluidic cartridge device. In some embodiments, the cartridgeshuttle is coupled to one or more translation or lateral movementprotocols to move the microfluidic cartridge device. For example, thecartridge shuttle may be coupled to a mechanically actuated translationstage, mechanical leadscrew assembly, mechanical slide device,mechanical lateral motion device, mechanically operated gearedtranslation device, a motor-actuated translation stage, leadscrewtranslation assembly, geared translation device, such as those employinga stepper motor, servo motor, brushless electric motor, brushed DCmotor, micro-step drive motor, high resolution stepper motor, amongother types of motors. Systems may also include a set of rails forpositioning the cartridge shuttle to facilitate lateral movement of thecartridge holder.

In some embodiments, systems further include a blank reference window toprovide a blank absorbance for use in calculating analyte concentration.The absorbance by the blank reference window is in certain embodiments,configured to be identical to absorbance by the sample chamber such thattransmission through the blank reference window can be used to correctfor absorption, scatter, etc. by the microfluidic cartridge whenpracticing the methods described herein. In certain embodiments, theblank reference window has an absorbance and transmission at the one ormore wavelengths of incident light which is substantially the same asthe capillary channel sample chamber. In other embodiments, the blankreference window scatters light at the one or more wavelengths which issubstantially the same as the capillary channel sample chamber. In yetother embodiments, the blank reference window has an absorbance,transmission and scatters light at the one or more incident wavelengthswhich is substantially the same as the capillary channel sample chamber.In still other embodiments, the blank reference window has an index ofrefraction which is the same as the capillary channel sample chamber.

The blank reference window integrated into the subject systems may beany convenient size and shape. For example, the blank reference windowmay be in the form of a square, circle, oval, rectangle, pentagon,hexagon, octagon or any other suitable polygon. In some embodiments, theblank reference window has a ratio of length to width which ranges from1 to 50, such as 3 to 25, such as from 4 to 10, such as from 5 to 8,including 15 to 20. In certain embodiments, the blank reference windowis a square and has a ratio of length to width of 1. The length of theblank reference window may vary, ranging from 1 mm to 50 mm, such as 2mm to 25 mm and including 5 mm to 20 mm. The width of the blankreference window may vary, ranging from 0.001 mm to 20 mm, such as from0.005 mm to 19 mm, such as from 0.01 mm to 18 mm, such as from 0.05 mmto 17 mm, such as from 0.1 mm to 15 mm, such as from 0.5 mm to 12.5 mm,such as 1 to 10 and including 3 to 5 mm. In some instances the height ofthe channel ranges from 5 μm to 500 μm, such as 10 μm to 150 μm andincluding 20 μm to 70 μm. In certain embodiments, the blank referencewindow has a width which is substantially the same as the width of thecapillary channel sample chamber. As described above, the slit-shapedbeam of light that is transmitted through the sample chamber iscollected and detected using one or photodetectors. In certainembodiments, systems include one or more objective lenses for collectinglight transmitted through the sample chamber. For example, the objectivelens may be a magnifying lens with a nominal magnification ranging from1.2 to 2.5, such as a nominal magnification of from 1.3 to 2.4, such asa nominal magnification of from 1.4 to 2.3, such as a nominalmagnification of from 1.5 to 2.2, such as a nominal magnification orfrom 1.6 to 2.1, including passing the transmitted light through amagnifying lens having a nominal magnification of from 1.7 to 2.0, forexample a nominal magnification of 1.7. In certain instances, theobjective lens is a magnifying achromatic doublet lens with a nominalmagnification of 1.7. Depending on the configuration of the lightsource, sample chamber and detector, properties of the objective lensmay vary. For example, the numerical aperture of the subject objectivelens may also vary, ranging from 0.01 to 1.7, such as from 0.05 to 1.6,such as from 0.1 to 1.5, such as from 0.2 to 1.4, such as from 0.3 to1.3, such as from 0.4 to 1.2, such as from 0.5 to 1.1 and including anumerical aperture ranging from 0.6 to 1.0. Likewise, the focal lengthof the objective lens varies, ranging from 10 mm to 20 mm, such as from10.5 mm to 19 mm, such as from 11 mm to 18 mm and including from 12 mmto 15 mm.

In some embodiments, the objective lens is coupled to an autofocusmodule for focusing the slit-shaped beam projection transmitted throughthe sample chamber onto the detector for detection. For example, asuitable autofocus module for focusing the slit-shaped beam projectiontransmitted through the sample may include, but is not limited, to thosedescribed in U.S. Pat. No. 6,441,894, filed on Oct. 29, 1999, thedisclosure of which is herein incorporated by reference.

Systems of the present disclosure may also include one or morewavelength separators. As discussed above, a “wavelength separator” isconfigured to separate polychromatic light into component wavelengthssuch that each wavelength may be suitably detected. Examples of suitablewavelength separators in the subject systems may include but are notlimited to colored glass, bandpass filters, interference filters,dichroic mirrors, diffraction gratings, monochromators and combinationsthereof, among other wavelength separating protocols. Depending on thelight source and sample being assayed, systems may include one or morewavelength separators, such as two or more, such as three or more, suchas four or more, such as five or more and including 10 or morewavelength separators. In one example, systems include two or morebandpass filters. In another example, systems include two or morebandpass filters and a diffraction grating. In yet another example,systems include a plurality of bandpass filters and a monochromator. Incertain embodiments, systems include a plurality of bandpass filters anddiffraction gratings configured into a filter wheel setup. Where systemsinclude two or more wavelength separators, the wavelength separators maybe utilized individually or in series to separate polychromatic lightinto component wavelengths. In some embodiments, wavelength separatorsare arranged in series. In other embodiments, wavelength separators arearranged individually such that one or more measurements are conductedto collect the desired absorbance data using each of the wavelengthseparators.

In some embodiments, systems include one or more diffraction gratings.Diffraction gratings of interest may include, but are not limited totransmission, dispersive or reflective diffraction gratings. Suitablespacings of the diffraction grating may vary depending on theconfiguration of the light source, slit projection module, samplechamber, objective lens, ranging from 0.01 μm to 10 μm, such as from0.025 μm to 7.5 μm, such as from 0.5 μm to 5 μm, such as from 0.75 μm to4 μm, such as from 1 μm to 3.5 μm and including from 1.5 μm to 3.5 μm.

In some embodiments, systems include one or more optical filters. Incertain instances, systems include bandpass filters having minimumbandwidths ranging from 2 nm to 100 nm, such as from 3 nm to 95 nm, suchas from 5 nm to 95 nm, such as from 10 nm to 90 nm, such as from 12 nmto 85 nm, such as from 15 nm to 80 nm and including bandpass filtershaving minimum bandwidths ranging from 20 nm to 50 nm. For example,systems may include one or more bandpass filters which selectively passwavelengths in the ranges of: 498 nm-510 nm; 500 nm-600 nm; 500 nm-520nm; 540 nm-550 nm; 545 nm-555 nm; 550 nm-570 nm; 550 nm-580 nm; 560nm-590 nm; 575 nm-595 nm; 580 nm-590 nm; 600 nm-700 nm; 600 nm-630 nm;650 nm-750 nm; 750 nm-850 nm; 810 nm-830 nm; 815 nm-825 nm or anycombination thereof.

In certain instances, systems include one or more bandpass filters whichselectively pass wavelengths ranging from 500 nm-520 nm and from 650nm-750 nm. In other instances, systems include one or more bandpassfilters which selectively pass wavelengths ranging from 540 nm-560 nmand from 650 nm-750 nm. In yet other instances, systems include one ormore bandpass filters which selectively pass wavelengths ranging from560 nm-590 nm and from 650 nm-750 nm. In still other instances, systemsinclude one or more bandpass filters which selectively pass wavelengthsranging from 500 nm-520 nm; 560 nm-590 nm and from 650 nm-750 nm.

Systems of the present disclosure also include one or more detectors.Examples of suitable detectors may include, but are not limited tooptical sensor or photodetectors, such as active-pixel sensors (APSs),avalanche photodiode, image sensors, charge-coupled devices (CCDs),intensified charge-coupled devices (ICCDs), light emitting diodes,photon counters, bolometers, pyroelectric detectors, photoresistors,photovoltaic cells, photodiodes, photomultiplier tubes,phototransistors, quantum dot photoconductors or photodiodes andcombinations thereof, among other photodetectors. In certainembodiments, the transmitted light is measured with a charge-coupleddevice (CCD). Where the transmitted light is measured with a CCD, theactive detecting surface area of the CCD may vary, such as from 0.01 cm²to 10 cm², such as from 0.05 cm² to 9 cm², such as from, such as from0.1 cm² to 8 cm², such as from 0.5 cm² to 7 cm² and including from 1 cm²to 5 cm².

In embodiments of the present disclosure, detectors of interest areconfigured to measure light transmitted through the sample chamber atone or more wavelengths, such as at 2 or more wavelengths, such as at 5or more different wavelengths, such as at 10 or more differentwavelengths, such as at 25 or more different wavelengths, such as at 50or more different wavelengths, such as at 100 or more differentwavelengths, such as at 200 or more different wavelengths, such as at300 or more different wavelengths and including measuring the lighttransmitted through the sample chamber at 400 or more differentwavelengths.

In some embodiments, detectors of interest are configured to measurelight transmitted through the sample chamber over a range of wavelengths(e.g., 400 nm-800 nm; 495 nm-525 nm; 800 nm-835 nm, etc.). For example,systems may include one or more detectors configured to measure lighttransmitted through the sample chamber over one or more of thewavelength ranges of: 400 nm-800 nm; 498 nm-510 nm; 500 nm-600 nm; 500nm-700 nm; 500 nm-520 nm; 540 nm-550 nm; 545 nm-555 nm; 550 nm-570 nm;550 nm-580 nm; 560 nm-590 nm; 575 nm-595 nm; 580 nm-590 nm; 600 nm-700nm; 600 nm-630 nm; 650 nm-750 nm; 750 nm-850 nm; 810 nm-830 nm; 815nm-825 nm and any combinations thereof. In certain instances, thedetector is configured to measure transmitted light over the wavelengthsranging from 400 nm-800 nm. In other instances, the detector isconfigured to measure transmitted light over the wavelengths rangingfrom 500 nm-520 nm and 650 nm-750 nm. In other instances, the detectoris configured to measure transmitted light over wavelengths ranging from540 nm-560 nm and 650 nm-750 nm. In yet other instances, the detector isconfigured to measure transmitted light over wavelengths ranging from560 nm-590 nm and 650 nm-750 nm. In still other instances, the detectoris configured to measure transmitted light over wavelengths ranging from500 nm-520 nm, 560 nm-590 nm, and 650-750 nm.

In certain embodiments, detectors of interest are configured to collectspectra of light over a range of wavelengths. For example, systems mayinclude one or more detectors configured to collect spectra of lightover one or more of the wavelength ranges of: 400 nm-800 nm; 498 nm-510nm; 500 nm-600 nm; 500 nm-700 nm; 500 nm-520 nm; 540 nm-550 nm; 545nm-555 nm; 550 nm-570 nm; 550 nm-580 nm; 560 nm-590 nm; 575 nm-595 nm;580 nm-590 nm; 600 nm-700 nm; 600 nm-630 nm; 650 nm-750 nm; 750 nm-850nm; 810 nm-830 nm; 815 nm-825 nm and any combinations thereof. Incertain instances, the detector is configured to collect spectra oflight having wavelengths ranging from 400 nm-800 nm. In other instances,the detector is configured to collect spectra of light havingwavelengths ranging from 500 nm-700 nm.

In yet other embodiments, detectors of interest are configured tomeasure light at one or more specific wavelengths. For example, systemsmay include one or more detectors configured to measure light at one ormore of 504 nm, 506 nm, 514 nm, 532 nm, 543 nm, 548 nm, 550 nm, 561 nm,568 nm, 579 nm, 580 nm, 585 nm, 586 nm, 675 nm, 710 nm, 808 nm, 815 nm,830 nm and any combinations thereof. In certain instances, the detectoris configured to measure light at 548 nm. In other instances, thedetector is configured to measure light at 675 nm. In other instances,the detector is configured to measure light at 830 nm. In yet otherinstances, the detector is configured to measure light at 548 nm and 675nm. In still other embodiments, the detector is configured to measure at548 nm, 675 nm and 830 nm.

In embodiments, the detector may be configured to measure lightcontinuously or in discrete intervals. In some instances, detectors ofinterest are configured to measure light continuously. In otherinstances, detectors of interest are configured to take measurements indiscrete intervals, such as measuring light every 0.001 millsecond,every 0.01 millsecond, every 0.1 millsecond, every 1 millsecond, every10 millseconds, every 100 millseconds and including every 1000millseconds, or some other interval.

Embodiments of the subject systems may also include one or more opticalcomponents as desired to provide any desired configuration. For example,systems may having each component (i.e., light source, slit projectionmodule, sample chamber, objective lens, wavelength separator anddetector) in an “in-line” configuration where light emitted from thelight source travels through each component without substantialdiversion form its inline pathway. Where desired, one or more mirrors,beam splitters or other types of light diversion components may be usedto divert light along a different path or to separate light intoseparate beams for detection.

FIG. 2 a depicts a side configuration of the subject systems accordingto one embodiment. A light source (201) provides light emitting one ormore wavelengths of light through a slit projection module whichincludes a slit (202 a) for narrowing the beam of light producing aslit-shaped beam and an objective lens (202 b) for focusing andde-magnifying the slit-shaped beam. The slit-shaped beam illuminates asample chamber (203) where analyte in the sample absorbs light and aremaining portion of the light is transmitted and collected by anobjective lens (204) which is configured to focus the light onto awavelength separator (e.g., diffraction grating 205) that spatiallyseparates the light into its component wavelengths. The spatiallyseparated light is then detected by a detector (e.g., CCD, 206).

FIG. 2 b depicts a top view of a configuration of the subject systemsaccording to another embodiment. A light source (207) provides lightemitting one or more wavelengths of light through a slit projectionmodule (208) which includes a slit and an objective lens for providing aslit-shaped beam. The slit-shaped beam illuminates a sample chamber(209) where analyte in the sample absorbs light and a remaining portionof the light is transmitted and collected by an objective lens (210) andprovided onto a wavelength separator (211) that spatially separates thelight into its component wavelengths. The spatially separated light isthen detected by a detector (212).

In some embodiments, systems are configured having one or more of thelight source, slit projection module, sample chamber, objective lens,wavelength separator and detector is in an offset position. By “offset”is meant that the geometric center of the system component is positionedat a point in space not along the central optical axis that connects thelight source and the detector. For example, in some embodiments, lightsource is in an offset position. In other embodiments, the slitprojection module is in an offset position. Where the slit projectionmodule includes a focusing lens, the focusing lens is, in certaininstances, in an offset position. In yet other embodiments, the samplechamber is in an offset position. In still other embodiments, theobjective lens for collecting and focusing transmitted light is in anoffset position. In still other embodiments, the wavelength separator isin an offset position. In certain embodiments, the detector is in anoffset position. In some instances, all of the light source, slitprojection module, sample chamber, objective lens, wavelength separatorand detector are in an offset position.

The offset distance may vary, as desired, ranging from 0.1 mm to 100 mm,such as from 0.2 mm to 95 mm, such as from 0.3 mm to 90 mm, such as from0.4 mm to 85 mm, such as from 0.5 mm to 80 mm, such as from 0.6 mm to 75mm, such as from 0.7 mm to 70 mm, such as from 0.8 mm to 65 mm, such asfrom 0.9 mm to 60 mm, such as from 1 mm to 55 mm, such as from 1.25 mmto 50 mm, such as from 1.5 mm to 45 mm, such as from 1.75 mm to 40 mm,such as from 2 mm to 30 mm, such as from 2.5 mm to 25 mm and includingfrom 1 mm to 20 mm.

For example, where the wavelength separator is a diffraction grating,the diffraction grating may be in an offset position to facilitatediffraction of desired wavelengths (e.g., 548 nm, 675 nm, etc.) for aspecific assay protocol. In another example, where the detector is a CCDdetector, the CCD detector may be in an offset position to facilitatedcollection of light (e.g., transmitted or emitted) at position on thedetector with calibrated pixels.

In certain embodiments, systems of interest include an integratedmicrofluidic sampling device having a capillary channel sample chamberwith a sample application site coupled to an inlet for inputting a fluidsample. Accordingly, in these embodiments, the subject systems are notconfigured to receive the microfluidic cartridge device described above,but instead are configured to receive the fluid sample directly, whichis subsequently removed following assay of the sample.

In embodiments, the integrated sampling device includes a capillarychannel sample chamber and a sample application site coupled to an inletfor inputting the fluid sample. The capillary channel sample chamber ofthe integrated microfluidic sampling device may have an elongatedstructure, such that it has a length that is longer than its width.While the ratio of length to width may vary, in some instances the ratioof length to width ranges from 2 to 5000, such as 3 to 2500, such asfrom 4 to 1000, such as from 5 to 500, such as from 6 to 100, such asfrom 10 to 50 and including 15 to 20. In some instances, the length ofthe channel ranges from 10 mm to 500 mm, such as 20 mm to 250 mm andincluding 50 mm to 75 mm. In some instances, the channels have amicrometer sized cross-sectional dimension, e.g., a longestcross-sectional dimension (e.g., diameter in the case of the tubularchannel) ranging from 0.1 mm to 20 mm, such as 1 mm to 10 mm andincluding 3 mm to 5 mm. In some instances the width of the channelranges from 0.001 mm to 20 mm, such as from 0.005 mm to 19 mm, such asfrom 0.01 mm to 18 mm, such as from 0.05 mm to 17 mm, such as from 0.1mm to 15 mm, such as from 0.5 mm to 12.5 mm, such as 1 to 10 andincluding 3 to 5 mm. In some instances the height of the channel rangesfrom 5 μm to 500 μm, such as 10 μm to 150 μm and including 20 μm to 70μm. Likewise, the capillary channel may have a cross sectional shapesuch as rectilinear cross sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, etc., as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion, etc.

The sample application site of the integrated microfluidic samplingdevice is a structure configured to receive a sample having a volumeranging from 5 μL to 100 μL, such as 10 μl_(—) to 50 μl_(—) andincluding 20 μl_(—) to 30 μL. The sample application site can have anyconvenient shape, so long as it provides for fluid access, eitherdirectly or through an intervening component(s) that provides forfluidic communication, to the capillary channel.

The inlet of the integrated microfluidic sampling device is in fluidiccommunication with sample application site and the capillary channelsample chamber and may be any suitable shape, where cross-sectionalshapes of inlets of interest include, but are not limited to:rectilinear cross sectional shapes, e.g., squares, rectangles,trapezoids, triangles, hexagons, etc., curvilinear cross-sectionalshapes, e.g., circles, ovals, etc., as well as irregular shapes, e.g., aparabolic bottom portion coupled to a planar top portion, etc. Dependingon the shape of the inlet, the sample inlet may have an opening sizewhich varies, ranging from 0.1 mm² to 100 mm², such as 1 mm² to 75 mm²and including 5 mm² to 50 mm².

In some instances, the integrated microfluidic sampling device includesa mixing chamber positioned in the fluidic path between the sampleapplication site and the capillary channel sample chamber that isconfigured to combine sample which has been applied to the sampleapplication and is flowing through the capillary channel with one ormore reagents.

In some instances, the mixing chamber of the integrated microfluidicsampling device includes a contacting structure that provides for highsurface area (e.g., porous disc) upon which one or more reagents may bepositioned, where in certain instances the high surface area structureis configured to filter or facilitate contact between one or morecomponents of the sample with reagents present in the mixing chamber. Incertain instances, the high surface area structure is configured to notfilter components of the sample and to simply facilitate contact betweenthe reagents and the sample flowing therethrough. For example, where thesample is a whole blood sample, the high surface area structure may beone that is configured not to impede the flow of any of the whole bloodcomponents, e.g., white blood cells, red blood cells, platelets, etc.,through the high surface area structure. In such instances, the highsurface area structure may have a porosity ranging from 20 to 80, suchas 30 to 70 and including 40 to 60. Suitable high surface area, porousmaterials for facilitating contact between sample and reagents include,but are not limited to, polymeric materials, glass materials, ceramicmaterials, metallic materials, etc. such as for example, polyethylene,polypropylene, polyvinylidine fluoride, and the like.

The reagents contained in the mixing chamber may, for example, includespecific binding members, enzymes, substrates, oxidizers, fluorescentmarkers, etc., such as those described below. Likewise, the amount ofreagent present in the mixing chamber or contacting structure of theintegrated microfluidic sampling device may vary, e.g., depending on theparticular type of assay for which the device is configured. In someinstances, the amount of a reagent is sufficient to provide for aconcentration of reagent in the sample following flow through the mixingchamber that ranges from 0.002 microgram/mL to 100 microgram/mL, such as0.02 microgram/mL to 10 microgram/mL and including 0.2 to 1microgram/mL. While the dry weight of a reagent present in the mixingchamber may vary, in some instances the dry weight ranges from 0.01 ngto 500 ng, such as 0.3 ng to 120 ng and including 3 ng to 12 ng.

As discussed above, where the subject systems include an integratedmicrofluidic sampling device, the sampling device is configured toreceive a fluid sample which is subsequently removed following assay ofthe sample. By “removed” is meant that no amount of the sample remainsin contact with the subject systems, including any of the capillarychannel sample chamber, sample application site, inlet, as well asmixing chamber. In other words, when the sample is removed, all tracesof the sample are cleared from the components of the system. In someembodiments, systems may further include one or more washing devices forcleaning the integrated microfluidic sampling device. For example, thewashing devices may include microconduits with or without spray nozzlesfor delivering wash buffer to clean the sampling device. In certainembodiments, these systems include a reservoir for storage of one ormore wash buffers.

In some instances, the subject systems may include one or morecomponents for reading or interrogating a unique identifier (e.g.,barcode, serial number) on the microfluidic cartridge, where identifiersof interest may provide information about the device, e.g., theparticular assay for which it is configured, manufacturing lot number,etc., which identifiers may be unique identifiers. The identifiers may,in certain instances, provide information or characteristics about themicrofluidic cartridge, including but not limited to index of refractionof the sample channel, index of refraction of the blank referencewindow, sample channel dimensions including sample channel height,sample channel width, sample channel length, overall sample channeldepth, thickness of the sample channel walls. Likewise, the identifiersmay include information about the blank reference window, such as indexof refraction of the blank reference window, blank reference windowdimensions including blank reference window height, blank referencewindow width, blank reference window length, overall blank referencewindow, thickness of the blank reference window walls.

Any convenient identification reader or interrogation protocol may beemployed, including but not limited to a bar code reader, RFIDinterrogation systems, magnetic strip readers, tactile code identifiers,among other identification protocols.

In certain embodiments, the subject absorbance detection systems may becoupled to one or more fluorescence detection systems for interrogatingthe sample chamber for fluorescence. For example, fluorescence detectionsystems may include one or more light sources (e.g., broadband or narrowband light sources, such as those described above), optical module forcollecting and focusing emission light, wavelength separators forspatially separating the collected light to be detected and one or morephotosensors or photodetectors.

In certain embodiments, both the fluorescence detection systems and theabsorbance detection systems employ one or more common components asdescribed herein. For example, in some instances both fluorescencedetection systems and absorbance detection systems employ a commonobjective lens module for collecting and focusing light from the samplechamber (e.g., emitted light or transmitted light). In other instances,both fluorescence detection systems and absorbance detection systemsemploy a common wavelength separator apparatus (e.g., diffractiongrating, optical filters, filter wheel having one or more diffractiongratings and optical filters). In yet other instances, both fluorescencedetection systems and absorbance detection systems employ the samedetector for measuring light from the sample chamber.

Fluorescence imaging and digital processing systems which may be coupledto the subject systems, in certain instances, include systems forimaging samples in capillary channels such as those described in U.S.Pat. Nos. 8,248,597; 7,927,561 and 7,738,094 as well as those describedin co-pending U.S. patent application Ser. No. 13/590,114 filed Aug. 20,2012, the disclosures of which are herein incorporated by reference.

FIG. 3 depicts a configuration of the subject systems according toanother embodiment where an absorbance detection system is coupled to afluorescence detection system. As discussed above, the absorbancedetection systems includes a light source (301) emitting one or morewavelengths of light through a slit projection module (302) whichincludes a slit for narrowing the beam of light producing a slit-shapedbeam and an objective lens for focusing and de-magnifying theslit-shaped beam. The slit-shaped beam illuminates a sample chamber in amicrofluidic cartridge (303) where analyte in the sample absorbs lightand a remaining portion of the light is transmitted and collected by anobjective lens (304) which is configured to focus the light onto awavelength separator 305 (e.g., filter wheel having one or more opticalfilters and diffraction gratings) that spatially separates the lightinto its component wavelengths. The spatially separated light is thendetected by a detector (e.g., CCD, 306). For fluorescence detection, thesystem includes a second light source (307) which is positioned abovethe sample chamber to illuminate the sample chamber and fluorescenceproduced by the analytes is collected with objective lens (304),spatially separated using filter wheel 305 and detected by detector 306.In this embodiment, the fluorescence detection system and the absorbancedetection system employs a common objective lens (304) for collectinglight from the sample chamber (303), wavelength separator apparatus(305) and detector (306).

In some embodiments, systems of interest also include one or moreprocessors for processing assay data collected, a display, such as aliquid crystal display (LCD) for displaying raw data collected duringassay or processed results received from the processor, as well as inputdevices, such as buttons, a keyboard, mouse or touch-screen. Inaddition, systems may include one or more of a wired or wirelesscommunication protocols or an integrated printer to communicate theresults to one or more users. For example, systems may include one ormore computer system components, such as those as described in greaterdetail below.

FIG. 4 depicts an example of an assay reader system of interestaccording one embodiment. System includes a housing (400) having a frontside (400 a) and a back side (400 b). Front side (400 a) includes adisplay (401), such as a liquid crystal display (LCD) for displaying rawdata collected during assay or processed results received from theprocessor, an integrated printer (403) for communicating assay data tothe user, as well as a slot for inserting (404) microfluidic cartridgedevice into the assay reader system. The assay reader system alsoincludes a communication interface (405) to allow data communication(e.g., USB port) between the subject system and one or more otherexternal devices such as a computer terminal that is configured forsimilar complementary data communication. Back side (400 b) can includecable connections for power and data communication protocols (not shown)as well as cooling vents operably connected to cooling fans and heatsinks. Back side (400 b) can also include one or more lift handles formanually moving or carrying the assay reader system. As shown in FIG. 4,the subject systems can be configured to be a unitary, “all-in-one”-typesystem which all of the optics, electronics (described in greater detailbelow), display, communication protocols in a single housing. In certainembodiments, systems of interest are configured to be capable formovement (lifted or carried a distance of 50 meters or more) by a humanwithout machine assistance (e.g., with lift handles). As such, systemsof interest, in these embodiments, are 25 kg or less, such as 20 kg orless, such as 15 kg or less and including 10 kg or less, e.g., 5 kg orless, and may include, as desired, a built in handle or other structureto provide for ease of manipulation/transportation.

Microfluidic Cartridge Devices

Aspects of the present disclosure also include a microfluidic cartridgedevice that is configured to be received by certain systems describedherein. In embodiments, the microfluidic cartridge device includes acapillary channel sample chamber in fluid communication with a sampleapplication site coupled to an inlet for inputting the fluid sample. Theterm “microfluidic” is used herein in its conventional sense to refer toa device that is configured to control and manipulate fluidsgeometrically constrained to a small scale (e.g., sub-millimeter). Asthe devices include a capillary channel sample chamber, they include anelongated structure that is configured to provide for capillary flow ofliquid therethrough. In addition to the sample application site,capillary channel sample chamber and inlet, aspects of the microfluidicdevice includes a reagent mixing chamber in communication with thesample application site and inlet for contacting and mixing the fluidicsample with one or more reagents. In certain embodiments, the mixingchamber is configured as a porous disc containing reagents forcontacting with the sample.

In embodiments of the present disclosure, the capillary channel samplechamber is an elongated structure, such that it has a length that islonger than its width. While the ratio of length to width may vary, insome instances the ratio of length to width ranges from 2 to 5000, suchas 3 to 2500, such as from 4 to 1000, such as from 5 to 500, such asfrom 6 to 100, such as from 10 to 50 and including 15 to 20. In someinstances, the length of the channel ranges from 10 mm to 500 mm, suchas 20 mm to 250 mm and including 50 mm to 75 mm. In some instances, thechannels have a micrometer sized cross-sectional dimension, e.g., alongest cross-sectional dimension (e.g., diameter in the case of thetubular channel) ranging from 0.1 mm to 20 mm, such as 1 mm to 10 mm andincluding 3 mm to 5 mm. In some instances the width of the channelranges from 0.001 mm to 20 mm, such as from 0.005 mm to 19 mm, such asfrom 0.01 mm to 18 mm, such as from 0.05 mm to 17 mm, such as from 0.1mm to 15 mm, such as from 0.5 mm to 12.5 mm, such as 1 to 10 andincluding 3 to 5 mm. In some instances the height of the channel rangesfrom 5 μm to 500 μm, such as 10 μm to 150 μm and including 20 μm to 70μm.

The cross sectional shape of the capillary channels may vary, in someinstances, cross-sectional shapes of channels of interest include, butare not limited to: rectilinear cross sectional shapes, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion, etc.

Positioned at one end of capillary channel (i.e., the proximal end) is asample application site having a fluidic inlet for conveying the sampleinto the capillary channel sample chamber. The sample application siteis a site or location configured to receive a volume of sample, e.g., abiological sample, to be analyzed. In some instances, the sampleapplication site is a structure configured to receive a sample having avolume ranging from 5 μl_(—) to 100 μL, such as 10 μl_(—) to 50 μl_(—)and including 20 μl_(—) to 30 μl_(—) The sample application site canhave any convenient shape, so long as it provides for fluid access,either directly or through an intervening component(s) that provides forfluidic communication, to the capillary channel.

The sample application site is in communication with an inlet to one endof the capillary channel sample chamber. The sample application site maybe positioned along a side of the microfluidic device such that sampleapplied to the sample application site is drawn into the inlet of thecapillary channel sample chamber. The inlet for conveying the sampleinto the capillary channel sample chamber may be any suitable shape,where cross-sectional shapes of channels of interest include, but arenot limited to: rectilinear cross sectional shapes, e.g., squares,rectangles, trapezoids, triangles, hexagons, etc., curvilinearcross-sectional shapes, e.g., circles, ovals, etc., as well as irregularshapes, e.g., a parabolic bottom portion coupled to a planar topportion, etc. Depending on the shape of the inlet as well as thedimensions of the microfluidic cartridge, the sample inlet may have anopening size which varies, ranging from 0.1 mm² to 100 mm², such as 1mm² to 75 mm² and including 5 mm² to 50 mm².

In some embodiments, the fluid sample is preloaded onto the sampleapplication site and the preloaded microfluidic device is stored for apredetermined period of time before the sample is assayed. As such,systems of the present disclosure may also include one or more preloadedmicrofluidic cartridges. For example, the fluid sample may be preloadedonto the sample application site before assaying the sample for 0.001hours or more, such as 0.005 hours or more, such as 0.01 hours or more,such as 0.05 hours or more, such as 0.1 hours or more, such as 0.5 hoursor more, such as 1 hour or more, such as 2 hours or more, such as 4hours or more, such as 8 hours or more, such as 16 hours or more, suchas 24 hours or more, such as 48 hours or more, such as 72 hours or more,such as 96 hours or more, such as 120 hours or more, such as 144 hoursor more, such as 168 hours or more and storing the preloadedmicrofluidic device for 240 hours or more before assaying the sample orthe amount of storage time may range such as from 0.1 hours to 240hours, such as from 0.5 hours to 216 hours, such as from 1 hour to 192hours and including from 5 hours to 168 hours before assaying thesample.

In some embodiments, the microfluidic device includes a mixing chamberpositioned in the fluidic path between the sample application site andthe capillary channel sample chamber. By mixing chamber is meant an areaor location in the fluidic path that is configured to combine samplewhich has been applied to the sample application and is flowing to thecapillary channel with one or more reagents.

In some instances, the mixing chamber includes a contacting structurethat provides for high surface area (e.g., porous disc) upon which oneor more reagents may be positioned, where in certain instances the highsurface area structure is configured to filter or facilitate contactbetween one or more components of the sample with reagents present inthe mixing chamber. In certain instances, the high surface areastructure is configured to not filter components of the sample and tosimply facilitate contact between the reagents and the sample flowingtherethrough. For example, where the sample is a whole blood sample, thehigh surface area structure may be one that is configured not to impedethe flow of any of the whole blood components, e.g., white blood cells,red blood cells, platelets, etc., through the high surface areastructure. In such instances, the high surface area structure may have aporosity ranging from 20 to 80, such as 30 to 70 and including 40 to 60.Suitable high surface area, porous materials for facilitating contactbetween sample and reagents include, but are not limited to, polymericmaterials, glass materials, ceramic materials, metallic materials, etc.such as for example, polyethylene, polypropylene, polyvinylidinefluoride, and the like.

Present in the mixing chamber is one or more reagents, which reagentsmay be present on a surface of a high surface area structure whenpresent. A variety of different reagents may be present in the mixingchamber or domain of the device, depending on the particular assay forwhich the device is configured. Reagents of interest include labeledspecific binding members, enzymes, substrates, oxidizers, etc., amongothers. In certain embodiments, the one or more reagents in the mixingchamber include a labeled specific binding member. For example, thelabeled specific binding member may include a specific binding domainand a label domain. The terms “specific binding,” “specifically binds,”and the like, refer to the preferential binding of a domain (e.g., onebinding pair member to the other binding pair member of the same bindingpair) relative to other molecules or moieties in a solution or reactionmixture. The specific binding domain may bind (e.g., covalently ornon-covalently) to a specific epitope of an analyte of interest. Incertain aspects, specific binding domain non-covalently binds to atarget. In such instances, the specific binding domain association withthe binding target (e.g., cell surface marker) may be characterized by aKD (dissociation constant) of 10-5 M or less, 10-6 M or less, such as10-7 M or less, including 10-8 M or less, e.g., 10-9 M or less, 10-10 Mor less, 10-11 M or less, 10-12 M or less, 10-13 M or less, 10-14 M orless, 10-15 M or less, including 10-16 M or less.

A variety of different types of specific binding domains may be employedas the capture ligands. Specific binding domains of interest include,but are not limited to, antibody binding agents, proteins, peptides,haptens, nucleic acids, etc. The term “antibody binding agent” as usedherein includes polyclonal or monoclonal antibodies or fragments thatare sufficient to bind to an analyte of interest. The antibody fragmentscan be, for example, monomeric Fab fragments, monomeric Fab′ fragments,or dimeric F(ab)′2 fragments. Also within the scope of the term“antibody binding agent” are molecules produced by antibody engineering,such as single-chain antibody molecules (scFv) or humanized or chimericantibodies produced from monoclonal antibodies by replacement of theconstant regions of the heavy and light chains to produce chimericantibodies or replacement of both the constant regions and the frameworkportions of the variable regions to produce humanized antibodies. Incertain embodiments, reagents of interest include, CD4-PECy5, CD3-APC,CD45RA-APC, CD14-PE.

The label domain may be detectible based on, for example, fluorescenceemission maxima, fluorescence polarization, fluorescence lifetime, lightscatter, mass, molecular mass, or combinations thereof. In certainaspects, the label domain may be a fluorophore (i.e., a fluorescentlabel, fluorescent dye, etc.). Fluorophores can be selected from any ofthe many dyes suitable for use in analytical applications (e.g., flowcytometry, imaging, etc.). A large number of dyes are commerciallyavailable from a variety of sources, such as, for example, MolecularProbes (Eugene, Oreg.) and Exciton (Dayton, Ohio). Examples offluorophores that may be incorporated into the microparticles include,but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives such as acridine, acridine orange, acrindine yellow,acridine red, and acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine andderivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylaminocoumarin; diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein,and QFITC (XRITC); fluorescamine; IR144; IR1446; Green FluorescentProtein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™;Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate;4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin;o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G),4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride,rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolicacid and terbium chelate derivatives; xanthene; or combinations thereof.Other fluorophores or combinations thereof known to those skilled in theart may also be used, for example those available from Molecular Probes(Eugene, Oreg.) and Exciton (Dayton, Ohio). The fluorescent label may bedistinguishable based on fluorescence emission maxima, and optionallyfurther based on light scatter or extinction.

The amount of reagent present in the mixing chamber or contactingstructure may vary, e.g., depending on the particular type of assay forwhich the device is configured. In some instances, the amount of areagent is sufficient to provide for a concentration of reagent in thesample following flow through the mixing chamber that ranges from 0.002microgram/mL to 100 microgram/mL, such as 0.02 microgram/mL to 10microgram/mL and including 0.2 to 1 microgram/mL. While the dry weightof a reagent present in the mixing chamber may vary, in some instancesthe dry weight ranges from 0.01 ng to 500 ng, such as 0.3 ng to 120 ngand including 3 ng to 12 ng.

In some instances, the device may include an analyte specific capturedomain. An analyte specific capture domain is a domain or region of thecapillary channel from which a result may be read during use of thedevice. The analyte specific capture domain is positioned at somedistance downstream from the sample application site of the device. By“downstream” is meant the direction that the sample flows by capillaryaction, i.e., the direction of fluid flow from the sample applicationsite. The total distance fluid flows between the sample receiving regionand the detection region may vary, ranging in some instances from 2 cmto 500 cm, such as 10 cm to 100 cm and including 20 cm to 50 cm.

The analyte specific capture domain is a region that includes an amountof a capture probe, also referred to herein as a “detection captureprobe.” A detection capture probe is immobilized in the analyte specificcapture domain and specifically binds to target molecule of interest,e.g., an analyte, a control molecule, etc. The size of the detectioncapture probe region may vary, and in some instances the capture proberegion can have an area ranging from 0.01 cm² to 0.5 cm², such as 0.05cm² to 0.1 cm² and including 0.1 cm² to 0.2 cm². An analyte specificcapture domain can have a variety of different configurations, where theconfiguration can be random or the configuration can have a specificshape such as a line, circle, square, or more complex shape, such as across-shape, as desired. A given analyte specific capture domain caninclude a single capture probe or two or more different capture probes,where each of the two or more different capture probes, where when thedetection region includes two or more capture probes, the capture probescan be distinct from each other (i.e., bind to different targetmolecules), as desired.

In some embodiments an analyte specific capture domain may be providedthat includes particles displaying a specific binding member(s) for atarget molecule(s), e.g., an analyte(s) of interest, a control orreference molecule, etc. For example, in some embodiments the device mayinclude an analyte specific capture domain comprised of capture beadsimmobilized on a convenient surface, e.g., the upper surface, of adomain of the capillary channel, e.g., a capillary chamber in thecapillary channel, e.g., as described in PCT Application Serial No.PCT/US2012/065683 filed on Nov. 16, 2012 and hereby incorporated byreference. The capture beads may be coated with a binding reagent thatspecifically binds to the analyte of interest. In some embodiments, thecapture beads are coated with an antigen to which the antibody ofinterest specifically binds. In such instances, a fluorescently labeledreagent for detection may be added that specifically binds to theanalyte, enabling the detection of the captured analyte by itsfluorescence emissions. The capture beads may be immobilized to a spoton the upper surface of the capillary chamber through any suitablemeans. In some instances, beads stayed localized in the spot by passiveinteractions between the beads and the capillary chamber surface, butcovalent binding can be used, as desired.

Capture beads coated with different antigens can be localized indifferent spots within the capillary chamber to enable the multiplexeddetection of multiple analytes. Alternatively, capture beads coated withdifferent antigens can be distinguishably labeled using fluorescent dyesthat are distinguishable from each other and from the dye-labeleddetection reagents that are used to measure the captured analytes. Inthis manner, the beads can be immobilized in the same spot, butdistinguished by their fluorescent emissions. In other embodiments,labeling reagents may be disposed at an analyte specific capture domaindisposed at the sample application site and labeled sample may flow to areaction chamber in the capillary channel for detection.

In some instances, the microfluidic cartridge may include a qualitycontrol domain in the capillary channel, e.g., positioned near the endof the channel furthest from the sample application site. The qualitycontrol channel may vary, and may for example include a capture member,e.g., antibody, specific for a labeled reagent, etc., such as describedin greater detail below, e.g., to provide a confirmation that sampleflows through the device during a given assay.

In some instances, the microfluidic cartridge may include one or moreidentifiers, which identifiers may provide information about the device,e.g., the particular assay for which it is configured, manufacturing lotnumber, etc., which identifiers may be unique identifiers. Theidentifiers may be human and/or machine readable, e.g., may be text(e.g., serial numbers) or a bar code, as desired. The identifiers may,in certain instances, provide information or characteristics about themicrofluidic cartridge, including but not limited to index of refractionof the sample channel, index of refraction of the blank referencewindow, sample channel dimensions including sample channel height,sample channel width, sample channel length, overall sample channeldepth, thickness of the sample channel walls. Likewise, the identifiersmay include information about the blank reference window, such as indexof refraction of the blank reference window, blank reference windowdimensions including blank reference window height, blank referencewindow width, blank reference window length, overall blank referencewindow, thickness of the blank reference window walls.

In some embodiments, the microfluidic cartridge further includes a blankreference window which is also interrogated by the slit projectionmodule to provide a blank absorbance for use in calculating analyteconcentration. The absorbance by the blank reference window is incertain embodiments, configured to be identical to absorbance by thesample chamber such that transmission through the blank reference windowcan be used to correct for absorption, scatter, etc. by the microfluidiccartridge when practicing the methods described herein. In certainembodiments, the blank reference window has an absorbance andtransmission at the one or more wavelengths of incident light which issubstantially the same as the capillary channel sample chamber. In otherembodiments, the blank reference window scatters light at the one ormore wavelengths which is substantially the same as the capillarychannel sample chamber. In yet other embodiments, the blank referencewindow has an absorbance, transmission and scatters light at the one ormore incident wavelengths which is substantially the same as thecapillary channel sample chamber. In still other embodiments, the blankreference window has the same index of refraction as the capillarychannel sample chamber.

The blank reference window may be any convenient size and shape. Forexample, the blank reference window may be in the form of a square,circle, oval, rectangle, pentagon, hexagon, octagon or any othersuitable polygon. In some embodiments, the blank reference window has aratio of length to width which ranges from 1 to 50, such as 3 to 25,such as from 4 to 10, such as from 5 to 8, including 15 to 20. Incertain embodiments, the blank reference window is a square and has aratio of length to width of 1. The length of the blank reference windowmay vary, ranging from 1 mm to 50 mm, such as 2 mm to 25 mm andincluding 5 mm to 20 mm. The width of the blank reference window mayvary, ranging from 0.001 mm to 20 mm, such as from 0.005 mm to 19 mm,such as from 0.01 mm to 18 mm, such as from 0.05 mm to 17 mm, such asfrom 0.1 mm to 15 mm, such as from 0.5 mm to 12.5 mm, such as 1 to 10and including 3 to 5 mm. In some instances the height of the channelranges from 5 μm to 500 μm, such as 10 μm to 150 μm and including 20 μmto 70 μm. In certain embodiments, the blank reference window has a widthwhich is substantially the same as the width of the capillary channelsample chamber.

The blank reference window may be positioned at any convenient locationon the microfluidic cartridge. In certain embodiments, the blankreference window is positioned along the same axis as the capillarychannel sample chamber. For example, the blank reference window may bepositioned along the same axis as the capillary channel sample chamberat a position that is 1 mm away or more from the capillary channelsample chamber, such as 2 mm or more, such as 3 mm or more, such as 4 mmor more, such as 5 mm or more and including 10 mm or more away from thecapillary channel sample chamber.

FIG. 5 illustrates one example of a microfluidic cartridge having amicrofluidic sample chamber for absorbance measurement (501) and areference window to provide for a blank during absorbance measurement(502).

One example of a suitable microfluidic cartridge which may be receivedinto the systems described herein may include, but are not limited tothose described in copending U.S. patent application Ser. No. 14/152,954filed on Jan. 10, 2014, the disclosure of which is herein incorporatedby reference.

Computer-Controlled Systems

Aspects of the present disclosure further include computer controlledsystems for practicing the subject methods, where the systems furtherinclude one or more computers for automation or semi-automation of asystem for practicing methods described herein. In certain embodiments,systems include a computer having a computer readable storage mediumwith a computer program stored thereon, where the computer program whenloaded on the computer includes algorithm illuminating a sample in asample chamber with a light source; algorithm for moving a slitprojection module along a length of the sample chamber; algorithm fordetecting light transmitted through the sample chamber, algorithm forcalculating absorbance of light at one or more wavelengths using thedetected transmitted light and algorithm for calculating concentrationof an analyte based on the absorbance determined from the transmittedlight.

In some embodiments, systems include a computer program that includesalgorithm for calculating absorbance of light at one or more wavelengthsbased on transmitted light detected by the detector. Absorbance of lightby the target analyte is determined by inputting transmittance data fromthe detector into a processor which applies the Beer-Lambert Law tocalculate absorbance at a given wavelength:

Absorbance (λ)=−Log₁₀(I/I ₀)

where I is the intensity of the light transmitted through the samplechamber and I₀ is the intensity of incident light used to interrogatethe sample.

Systems also include a computer program that includes algorithm forcalculating concentration of the analyte based on calculated absorbanceat one or more wavelengths. The concentration of analyte, in certainembodiments, is calculated by inputting absorbance values calculatedbased on transmittance data into a processor which applies the formula:

Absorbance (λ)=[molar absorptivity_(λ)]×[concentration]×[pathlength].

In some embodiments, systems include algorithm for calculatingabsorbance of the analyte while accounting for scatter by the sample.The absorbance by the analyte while accounting for scatter by the sampleis determined, in certain instances, by inputting absorbance valuescalculated based on transmittance data into a processor which appliesthe formula:

Concentration_(analyte) =A*(Abs_(λ1))+B*(Abs_(λ2))+C,

where A, B, and C are coefficients which depend on the wavelengthsinterrogated and analytes being measured. In embodiments, the value of Amay vary, in certain instances, ranging from 20 g/dL to 60 g/dL, such asfrom 25 g/dL to 57.5 g/dL, such as from 30 g/dL to 55 g/dL, such as from35 g/dL to 50 g/dL and including from 37.5 g/dL to 45 g/dL. The value ofB may also vary, in certain instances, ranging from 0.01 g/dL to 5 g/dL,such as from 0.05 g/dL to 4.5 g/dL, such as from 0.1 g/dL to 4 g/dL,such as from 0.25 g/dL to 3.5 g/dL, such as from 0.5 g/dL to 3 g/dL andincluding from 0.5 g/dL to 2 g/dL. Likewise, the value of C may alsovary, ranging from 0.01 g/dL to 2 g/dL, such as from 0.025 g/dL to 1.75g/dL, such as from 0.05 g/dL to 1.5 g/dL, such as from 0.1 g/dL to 1.25g/dL and including from 0.25 g/dL to 2 g/dL.

For example, in certain instances systems are configured to calculatethe concentration of hemoglobin in whole blood while accounting forscatter. In these instances, systems include algorithm for calculatingabsorbance of hemoglobin in whole blood while accounting for scatter bythe whole blood sample. The system includes a computer program withalgorithm for choosing a first wavelength and second wavelength tointerrogate the sample. In these embodiments, the computer algorithmincludes choosing a first wavelength where hemoglobin has a high molarabsorptivity, which may be an isosbestic point for hemoglobin with oneor more of oxyhemoglobin, carboxyhemoglobin, methemoglobin,sulfo-hemoglobin, azide-methemoglobin and cyano-methemoglobin, such as aisosbestic point for hemoglobin and oxyhemoglobin or a triple isosbesticpoint for hemoglobin, oxyhemoglobin and carboxyhemoglobin. For example,the first wavelength is, in certain instances, 506 nm, 548 nm, 569 nm,579 nm, 585 nm or 586 nm. The computer algorithm also includes choosinga second wavelength to account for scatter. In some instances, thecomputer algorithm includes choosing a second wavelength is anisosbestic point for hemoglobin with one or more of oxyhemoglobin,carboxyhemoglobin, methemoglobin, sulfo-hemoglobin, azide-methemoglobinand cyano-methemoglobin, such as a isosbestic point for hemoglobin andoxyhemoglobin or a triple isosbestic point for hemoglobin, oxyhemoglobinand carboxyhemoglobin. For example, a second wavelength is, in certaininstances, 650 nm, 675 nm, 710 nm, 785 nm, 808 nm, 815 nm or 830 nm.

For example, in certain embodiments systems include a computer programthat includes algorithm for choosing a first wavelength of 548 nm and asecond wavelength of 675 nm and determining the concentration ofhemoglobin in whole blood while accounting for scatter in the wholeblood sample by: 1) inputting transmittance data in to a processorapplying the Beer Lambert Law; and 2) inputting the calculatedabsorbance values into a processor which applies the formula:

Concentration_(Hb) =A*(Abs_(548 nm))+B*(Abs_(675 nm))+C,

where the value of A for a whole blood sample ranges from 20 g/dL to 60g/dL, such as from 25 g/dL to 57.5 g/dL, such as from 30 g/dL to 55g/dL, such as from 35 g/dL to 50 g/dL and including from 37.5 g/dL to 45g/dL; the value of B for a whole blood sample ranges from 0.01 g/dL to 5g/dL, such as from 0.05 g/dL to 4.5 g/dL, such as from 0.1 g/dL to 4g/dL, such as from 0.25 g/dL to 3.5 g/dL, such as from 0.5 g/dL to 3g/dL and including from 0.5 g/dL to 2 g/dL and where the value of C of awhole blood sample ranges from 0.01 g/dL to 2 g/dL, such as from 0.025g/dL to 1.75 g/dL, such as from 0.05 g/dL to 1.5 g/dL, such as from 0.1g/dL to 1.25 g/dL and including from 0.25 g/dL to 2 g/dL.

In embodiments, the system includes an input module, a processing moduleand an output module. In some embodiments, the subject systems mayinclude an input module such that parameters or information about eachfluidic sample, intensity and wavelengths (discrete or ranges) of theapplied light source, amplitude of movement by the slit projectionmodule, number of scans and movement by the slit projection module,duration of illumination by the light source, number of different lightsources, distance from light source to sample chamber, focal length ofobjective lens, parameters of the focusing module, path length of samplechamber, refractive index of sample, refractive index of sample chamber,number of wavelength separators, properties of wavelength separatorsincluding bandpass width, opacity, grating spacting and resolution aswell as properties and sensitivity of photodetectors.

The processing module includes memory having a plurality of instructionsfor performing the steps of the subject methods, such as illuminating asample in a sample chamber with a light source; moving a slit projectionmodule along a length of the sample chamber; detecting light transmittedthrough the sample chamber and calculating absorbance of light at one ormore predetermined wavelengths using the detected transmitted light.

After the processing module has performed one or more of the steps ofthe subject methods, an output module communicates the results (e.g.,absorbance of the analyte at one or more wavelengths) to the user, suchas by displaying on a monitor or by printing a report.

The subject systems may include both hardware and software components,where the hardware components may take the form of one or moreplatforms, e.g., in the form of servers, such that the functionalelements, i.e., those elements of the system that carry out specifictasks (such as managing input and output of information, processinginformation, etc.) of the system may be carried out by the execution ofsoftware applications on and across the one or more computer platformsrepresented of the system.

Systems may include a display and operator input device. Operator inputdevices may, for example, be a keyboard, mouse, or the like. Theprocessing module includes a processor which has access to a memoryhaving instructions stored thereon for performing the steps of thesubject methods, such as illuminating a sample in a sample chamber witha light source; moving a slit projection module along a length of thesample chamber; detecting light transmitted through the sample chamberand calculating absorbance of light at one or more predeterminedwavelengths using the detected transmitted light.

The processing module may include an operating system, a graphical userinterface (GUI) controller, a system memory, memory storage devices, andinput-output controllers, cache memory, a data backup unit, and manyother devices. The processor may be a commercially available processoror it may be one of other processors that are or will become available.The processor executes the operating system and the operating systeminterfaces with firmware and hardware in a well-known manner, andfacilitates the processor in coordinating and executing the functions ofvarious computer programs that may be written in a variety ofprogramming languages, such as Java, Perl, C++, other high level or lowlevel languages, as well as combinations thereof, as is known in theart. The operating system, typically in cooperation with the processor,coordinates and executes functions of the other components of thecomputer. The operating system also provides scheduling, input-outputcontrol, file and data management, memory management, and communicationcontrol and related services, all in accordance with known techniques.

The system memory may be any of a variety of known or future memorystorage devices. Examples include any commonly available random accessmemory (RAM), magnetic medium such as a resident hard disk or tape, anoptical medium such as a read and write compact disc, flash memorydevices, or other memory storage device. The memory storage device maybe any of a variety of known or future devices, including a compact diskdrive, a tape drive, a removable hard disk drive, or a diskette drive.Such types of memory storage devices typically read from, and/or writeto, a program storage medium (not shown) such as, respectively, acompact disk, magnetic tape, removable hard disk, or floppy diskette.Any of these program storage media, or others now in use or that maylater be developed, may be considered a computer program product. Aswill be appreciated, these program storage media typically store acomputer software program and/or data. Computer software programs, alsocalled computer control logic, typically are stored in system memoryand/or the program storage device used in conjunction with the memorystorage device.

In some embodiments, a computer program product is described comprisinga computer usable medium having control logic (computer softwareprogram, including program code) stored therein. The control logic, whenexecuted by the processor the computer, causes the processor to performfunctions described herein. In other embodiments, some functions areimplemented primarily in hardware using, for example, a hardware statemachine. Implementation of the hardware state machine so as to performthe functions described herein will be apparent to those skilled in therelevant arts.

Memory may be any suitable device in which the processor can store andretrieve data, such as magnetic, optical, or solid state storage devices(including magnetic or optical disks or tape or RAM, or any othersuitable device, either fixed or portable). The processor may include ageneral purpose digital microprocessor suitably programmed from acomputer readable medium carrying necessary program code. Programmingcan be provided remotely to processor through a communication channel,or previously saved in a computer program product such as memory or someother portable or fixed computer readable storage medium using any ofthose devices in connection with memory. For example, a magnetic oroptical disk may carry the programming, and can be read by a diskwriter/reader. Systems of the invention also include programming, e.g.,in the form of computer program products, algorithms for use inpracticing the methods as described above. Programming according to thepresent invention can be recorded on computer readable media, e.g., anymedium that can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM;portable flash drive; and hybrids of these categories such asmagnetic/optical storage media.

The processor may also have access to a communication channel tocommunicate with a user at a remote location. By remote location ismeant the user is not directly in contact with the system and relaysinput information to an input manager from an external device, such as aa computer connected to a Wide Area Network (“WAN”), telephone network,satellite network, or any other suitable communication channel,including a mobile telephone (i.e., smartphone).

In some embodiments, systems according to the present disclosure may beconfigured to include a communication interface. In some embodiments,the communication interface includes a receiver and/or transmitter forcommunicating with a network and/or another device. The communicationinterface can be configured for wired or wireless communication,including, but not limited to, radio frequency (RF) communication (e.g.,Radio-Frequency Identification (RFID), Zigbee communication protocols,WiFi, infrared, wireless Universal Serial Bus (USB), Ultra Wide Band(UWB), Bluetooth® communication protocols, and cellular communication,such as code division multiple access (CDMA) or Global System for Mobilecommunications (GSM). In one embodiment, the communication interface isconfigured to include one or more communication ports, e.g., physicalports or interfaces such as a USB port, an RS-232 port, or any othersuitable electrical connection port to allow data communication betweenthe subject systems and other external devices such as a computerterminal (for example, at a physician's office or in hospitalenvironment) that is configured for similar complementary datacommunication.

In one embodiment, the communication interface is configured forinfrared communication, Bluetooth® communication, or any other suitablewireless communication protocol to enable the subject systems tocommunicate with other devices such as computer terminals and/ornetworks, communication enabled mobile telephones, personal digitalassistants, or any other communication devices which the user may use inconjunction therewith, in managing the treatment of a health condition,such as HIV, AIDS or anemia.

In one embodiment, the communication interface is configured to providea connection for data transfer utilizing Internet Protocol (IP) througha cell phone network, Short Message Service (SMS), wireless connectionto a personal computer (PC) on a Local Area Network (LAN) which isconnected to the internet, or WiFi connection to the internet at a WiFihotspot.

In one embodiment, the subject systems are configured to wirelesslycommunicate with a server device via the communication interface, e.g.,using a common standard such as 802.11 or Bluetooth® RF protocol, or anIrDA infrared protocol. The server device may be another portabledevice, such as a smart phone, Personal Digital Assistant (PDA) ornotebook computer; or a larger device such as a desktop computer,appliance, etc. In some embodiments, the server device has a display,such as a liquid crystal display (LCD), as well as an input device, suchas buttons, a keyboard, mouse or touch-screen.

In some embodiments, the communication interface is configured toautomatically or semi-automatically communicate data stored in thesubject systems, e.g., in an optional data storage unit, with a networkor server device using one or more of the communication protocols and/ormechanisms described above.

Output controllers may include controllers for any of a variety of knowndisplay devices for presenting information to a user, whether a human ora machine, whether local or remote. If one of the display devicesprovides visual information, this information typically may be logicallyand/or physically organized as an array of picture elements. A graphicaluser interface (GUI) controller may include any of a variety of known orfuture software programs for providing graphical input and outputinterfaces between the system and a user, and for processing userinputs. The functional elements of the computer may communicate witheach other via system bus. Some of these communications may beaccomplished in alternative embodiments using network or other types ofremote communications. The output manager may also provide informationgenerated by the processing module to a user at a remote location, e.g.,over the Internet, phone or satellite network, in accordance with knowntechniques. The presentation of data by the output manager may beimplemented in accordance with a variety of known techniques. As someexamples, data may include SQL, HTML or XML documents, email or otherfiles, or data in other forms. The data may include Internet URLaddresses so that a user may retrieve additional SQL, HTML, XML, orother documents or data from remote sources. The one or more platformspresent in the subject systems may be any type of known computerplatform or a type to be developed in the future, although theytypically will be of a class of computer commonly referred to asservers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known orfuture type of cabling or other communication system including wirelesssystems, either networked or otherwise. They may be co-located or theymay be physically separated. Various operating systems may be employedon any of the computer platforms, possibly depending on the type and/ormake of computer platform chosen. Appropriate operating systems includeWindows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux,OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others.

Kits

Aspects of the invention further include kits, where kits include one ormore microfluidic assay cartridges. In some instances, the kits caninclude one or more assay components (e.g., labeled reagents, buffers,etc., such as described above). In some instances, the kits may furtherinclude a sample collection device, e.g., a lance or needle configuredto prick skin to obtain a whole blood sample, a pipette, etc., asdesired.

FIG. 6 shows an example of a kit having a microfluidic cartridgepackaged together with a lancet for obtaining whole blood. FIG. 7 showsan example of a collection of different types of kits, which in certainembodiments may be packaged together and provided in a box.

The various assay components of the kits may be present in separatecontainers, or some or all of them may be pre-combined into a reagentmixture. For example, in some instances, one or more components of thekit, e.g., the device, are present in a sealed pouch, e.g., a sterilefoil pouch or envelope.

In addition to the above components, the subject kits may furtherinclude (in certain embodiments) instructions for practicing the subjectmethods. These instructions may be present in the subject kits in avariety of forms, one or more of which may be present in the kit. Oneform in which these instructions may be present is as printedinformation on a suitable medium or substrate, e.g., a piece or piecesof paper on which the information is printed, in the packaging of thekit, in a package insert, and the like. Yet another form of theseinstructions is a computer readable medium, e.g., diskette, compact disk(CD), portable flash drive, and the like, on which the information hasbeen recorded. Yet another form of these instructions that may bepresent is a website address which may be used via the internet toaccess the information at a removed site.

Utility

Methods, systems, microfluidic cartridges and kits of the presentdisclosure find use in a variety of different applications and can beused to determine the presence and amount of an analyte in a largenumber of different sample types from a multitude of possible sources.Depending on the application and the desired output of the methodsdescribed herein, an analyte may be detected in a qualitative manner(“present” vs “absent”; “yes, above a predetermined threshold” vs “no,not above a predetermined threshold”; etc.) or a quantitative manner,e.g., as an amount in a sample (such as concentration in sample).

The subject methods and systems can be employed to characterize manytypes of analytes, in particular, analytes relevant to medical diagnosisor protocols for caring for a patient, including but not limited to:proteins (including both free proteins and proteins bound to surface ofa structure, such as a cell), nucleic acids, viral particles, and thelike. Further, samples can be from in vitro or in vivo sources, andsamples can be diagnostic samples.

In practicing methods of the invention, the samples can be obtained fromin vitro sources (e.g., extract from a laboratory grown cell culture) orfrom in vivo sources (e.g., a mammalian subject, a human subject, aresearch animal, etc.). In some embodiments, the sample is obtained froman in vitro source. In vitro sources include, but are not limited to,prokaryotic (e.g., bacterial) cell cultures, eukaryotic (e.g.,mammalian, fungal) cell cultures (e.g., cultures of established celllines, cultures of known or purchased cell lines, cultures ofimmortalized cell lines, cultures of primary cells, cultures oflaboratory yeast, etc.), tissue cultures, column chromatography eluants,cell lysates/extracts (e.g., protein-containing lysates/extracts,nucleic acid-containing lysates/extracts, etc.), viral packagingsupernatants, and the like. In some embodiments, the sample is obtainedfrom an in vivo source. In vivo sources include living multi-cellularorganisms and can yield diagnostic samples.

In some embodiments, the analyte is a diagnostic analyte. A “diagnosticanalyte” is an analyte from a sample that has been obtained from orderived from a living multi-cellular organism, e.g., mammal, in order tomake a diagnosis. In other words, the sample has been obtained todetermine the presence of one or more disease analytes in order todiagnose a disease or condition. Accordingly, the methods are diagnosticmethods. As the methods are “diagnostic methods,” they are methods thatdiagnose (i.e., determine the presence or absence of) a disease (e.g.,sickness, diabetes, etc.) or condition (e.g., pregnancy) in a livingorganism, such as a mammal (e.g., a human). As such, certain embodimentsof the present disclosure are methods that are employed to determinewhether a living subject has a given disease or condition (e.g.,diabetes). “Diagnostic methods” also include methods that determine theseverity or state of a given disease or condition.

In certain embodiments, the methods are methods of determining whetheran analyte is present in a diagnostic sample. As such, the methods aremethods of evaluating a sample in which the analyte of interest may ormay not be present. In some cases, it is unknown whether the analyte ispresent in the sample prior to performing the assay. In other instances,prior to performing the assay, it is unknown whether the analyte ispresent in the sample in an amount that is greater than (exceeds) apredetermined threshold amount. In such cases, the methods are methodsof evaluating a sample in which the analyte of interest may or may notbe present in an amount that is greater than (exceeds) a predeterminedthreshold.

Diagnostic samples include those obtained from in vivo sources (e.g., amammalian subject, a human subject, and the like.) and can includesamples obtained from tissues or cells of a subject (e.g., biopsies,tissue samples, whole blood, fractionated blood, hair, skin, and thelike). In some cases, cells, fluids, or tissues derived from a subjectare cultured, stored, or manipulated prior to evaluation and such asample can be considered a diagnostic sample if the results are used todetermine the presence, absence, state, or severity of a disease (e.g.,sickness, diabetes, etc.) or condition (e.g., pregnancy) in a livingorganism.

In some instances, a diagnostic sample is a tissue sample (e.g., wholeblood, fractionated blood, plasma, serum, saliva, and the like) or isobtained from a tissue sample (e.g., whole blood, fractionated blood,plasma, serum, saliva, skin, hair, and the like). An example of adiagnostic sample includes, but is not limited to cell and tissuecultures derived from a subject (and derivatives thereof, such assupernatants, lysates, and the like); tissue samples and body fluids;non-cellular samples (e.g., column eluants; acellular biomolecules suchas proteins, lipids, carbohydrates, nucleic acids; synthesis reactionmixtures; nucleic acid amplification reaction mixtures; in vitrobiochemical or enzymatic reactions or assay solutions; or products ofother in vitro and in vivo reactions, etc.); etc.

In some embodiments, the subject methods provide an assay forhemoglobin. As discussed above, hemoglobin may be present in any type ofdiagnostic sample, such as supernatants, lysates, buffered solution, aswell as in biological samples including whole blood. An amount of wholeblood is loaded into a sample chamber and illuminated through a slitprojection module with one or more light sources, with light transmittedthrough the whole blood sample in the sample chamber being collected andspatially separated into component wavelengths for detection. Dependingon the size of the whole blood sample, the sample chamber may be amicrofluidic capillary channel sample chamber. Hemoglobin absorbance canbe determined from the transmitted light at one or more wavelengths oralternatively, an entire spectrum of hemoglobin absorption may becalculated. Based on the absorbance at one or more wavelengths, thehemoglobin concentration in the whole blood sample can be determined inthese embodiments of the subject methods.

In certain other instances, the subject methods provide a reagent freehemoglobin assay. As discussed above, a reagent free assay is an assayof hemoglobin which employs no reagents to interact or visualizehemoglobin in the sample. As such, hemoglobin (including derivativessuch oxy-hemoglobin and carboxyhemoglobin) is assayed in its nativestate without reagent modification. In these instances, an unalteredwhole blood sample is loaded into a sample chamber and illuminated withone or more light sources through a slit projection module, with lighttransmitted through the whole blood sample in the sample chamber beingcollected and spatially separated into component wavelengths fordetection. Depending on the size of the whole blood sample, the samplechamber may be a microfluidic capillary channel sample chamber.Hemoglobin absorbance can be detected at one or more wavelengths oralternatively, an entire spectrum of hemoglobin absorption may becalculated. Based on the absorbance at one or more wavelengths, thehemoglobin concentration in the unaltered whole blood sample can bedetermined in these embodiments of the subject methods.

In certain other instances, the subject methods provide a hemoglobinassay on a sample also being assayed for one or more additionalanalytes, such as for example cell surface markers. In theseembodiments, one or more reagents, including specific binding members,enzymes, substrates, oxidizers as well as binding molecules coupled toone or more fluorescent markers are contacted with the whole blood andthe reagent-mixed whole blood sample is loaded into a sample chamber.The loaded sample chamber (such as a microfluidic capillary channelsample chamber) is illuminated with one or more light sources through aslit projection module, with light transmitted through the whole bloodsample in the sample chamber being collected and spatially separatedinto component wavelengths for detection. Hemoglobin absorbance can bedetected at one or more wavelengths or alternatively, an entire spectrumof hemoglobin absorption may be calculated. Based on the absorbance atone or more wavelengths, the hemoglobin concentration in thereagent-mixed whole blood sample can be determined in these embodimentsof the subject methods. In conjunction with assaying for hemoglobin inthe reagent-mixed sample, one or more additional analytes may beassayed. In some instances, the subject methods provide a fluorescenceassay performed in conjunction with the hemoglobin absorbance assay toassay for one or more cell surface markers binding to the one or morereagents mixed into the whole blood sample. In these instances, afluorescence light source illuminates the sample chamber loaded withreagent-mixed whole blood sample and fluorescence emission fromfluorescence tags bound to target analytes is collected and spatiallyseparated for detection.

In certain specific instances, the subject methods provide a hemoglobinassay on a sample for which is being fluorescence assayed for CD4 and %CD4. In these instances, the whole blood sample is applied to the sampleapplication site of a microfluidic cartridge having a capillary channelsample chamber. The applied sample is carried through the inlet of themicrofluidic capillary channel into a reagent mixing chamber having aporous disc for contacting the reagent mixture with the whole bloodsample. The reagent mixture, in these instances, includes dried storagestable reagents CD4-PECy5, CD3-APC, CD45RA-APC and CD14-PE. The reagentmixed whole blood sample is carried by capillary action through to thesample chamber where the sample chamber is illuminated for hemoglobinassay by two light sources, a broadband white light LED and anear-infrared LED through a slit projection module which is movedlaterally across the sample chamber. Light transmitted though the samplechamber is collected with an objective, magnifying lens and autofocusedonto a diffraction grating to spatially separate the transmitted lighton the surface of a CCD detector. The absorbance at two wavelengths, 548nm and 675 nm are determined and the total hemoglobin absorbanceaccounting for scatter is calculated to assay for hemoglobin.

The reagent mixed whole blood sample in the capillary channel samplechamber is also assayed for CD4 by detecting fluorescence by fluorescenttags in the reagent mixture. CD4 may be assayed for by illuminating thereagent mixed whole blood sample in the capillary channel sample chamberwith a light source and emission from the fluorescent tags in thereagent mixed whole blood sample is collected with a common objective,magnifying lens and autofocused onto the surface of the CDD detector.CD4 cell counting is then conducted by fluorescent image cytometry.

The subject methods can be employed with samples from a variety ofdifferent types of subjects. In some embodiments, a sample is from asubject within the class mammalia, including e.g., the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats),lagomorpha (e.g. rabbits) and primates (e.g., humans, chimpanzees, andmonkeys), and the like. In certain embodiments, the animals or hosts,i.e., subjects are humans.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentdisclosure has a wide variety of applications. Accordingly, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Those of skill in the art will readily recognizea variety of noncritical parameters that could be changed or modified toyield essentially similar results. Thus, the following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentinvention, and are not intended to limit the scope of what the inventorsregard as their invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (e.g.amounts, temperature, etc.) but some experimental errors and deviationsshould be accounted for.

Example 1

A droplet (˜25 μL) of whole blood sample is applied to the sampleapplication site of a microfluidic cartridge device having a mixingchamber containing dried storage stable CD4 reagents (e.g., CD4-PECy5,CD3-APC, CD45RA-APC and CD14-PE) which are hydrated when mixed with thewhole blood sample. The microfluidic cartridge device is allowed to situntil capillary action carries the reagent mixed sample into thecapillary channel sample chamber. The sample chamber is illuminated by abroad spectrum LED light source (one white light LED and one near-IRLED, sequentially illuminated) with a wavelength range of 500 nm to 850nm through a slit projection module having a slit and demagnifying lensto focus the slit-shaped beam at the surface of the sample chamber. Thesample chamber is moved in a back-and-forth motion to pass light throughthe sample chamber which is diffracted using a diffraction gratinghaving 300 μm spacings onto a CCD detector. FIG. 8 illustrates lightdetected by the CCD detector in a plot of pixel column with respect towavelength (801) where white pixels indicate detected light. The plot at801 is compressed into a 1-D spectrum at (802) by plotting each pixelcolumn with respect to wavelength to detect a spectrum of transmittedlight with respect to wavelength (803). Using the Beer-Lambert Law,absorbance is calculated at (804) to provide spectrum of absorbance bythe sample at (805). The concentration of hemoglobin can be calculatedbased on the determined absorbance and the reference blank obtainedduring light measurement through the blank reference window on themicrofluidic cartridge.

Example 2

A droplet (˜25 μL) of whole blood samples having either 25 g/L or 7 g/Lhemoglobin is applied to the sample application site of a microfluidiccartridge device having a mixing chamber containing dried storage stablereagents. After the reagent-mixed sample reaches the capillary channelsample chamber, the sample chamber is illuminated by a broad spectrumLED light source (one white light LED and one near-IR LED, sequentiallyilluminated) with a wavelength range of 500 nm to 850 nm through a slitprojection module having a slit and demagnifying lens to focus theslit-shaped beam at the surface of the sample chamber. The samplechamber is moved passing light through the sample chamber which isdiffracted using a diffraction grating onto a CCD detector. Pixel plotsfrom the CCD detector are compressed to a one-dimensional spectrum oftransmitted light with respect to wavelength. Using the Beer-LambertLaw, absorbance spectra were calculated from the spectra of transmittedlight. FIG. 9 a shows absorbance spectra of hemoglobin in whole blood ata concentration of 25 g/dL. FIG. 9 b shows absorbance spectra ofhemoglobin in whole blood at a concentration of 7 g/dL. The absorbanceat 569 nm was determined at each concentration of hemoglobin from theobtained spectra. The protocol was repeated with whole blood sampleshaving hemoglobin concentrations of 3 g/dL, 13 g/dL, 19 g/dL andabsorbance at 569 nm for each of the whole blood samples was plottedwith respect to concentration. FIG. 9 c illustrates a linearrelationship between hemoglobin concentration and absorbance at 569 nmindicating that measurements using the slit-projection module can beused over a wide range of hemoglobin concentrations.

Example 3

120 fresh HIV+ patient whole blood samples (both venipuncture andfingerstick samples) were analyzed using a Sysmex XS-1000i automatedhematology system to determine concentration of hemoglobin in the wholeblood samples. In conjunction, each sample was applied to the sampleapplication site of a separate microfluidic cartridge device having amixing chamber containing dried storage stable CD4 assay reagents. Thesample chamber of each of the 120 microfluidic cartridge devices wereilluminated by a broad spectrum LED light source (one white light LEDand one near-IR LED, sequentially illuminated) with a wavelength rangeof 500 nm to 850 nm through a slit projection module having a slit anddemagnifying lens to focus the slit-shaped beam at the surface of thesample chamber. The sample chamber is moved through the slit-shaped beampassing light through the sample chamber which is diffracted using adiffraction grating onto a CCD detector. Pixel plots from the CCDdetector are compressed to a one-dimensional spectrum of transmittedlight with respect to wavelength. Using the Beer-Lambert Law, absorbancespectra were calculated from the spectra of transmitted light.Concentration of hemoglobin for each sample was calculated usingabsorbance at 548 nm and corrected for scatter by measuring absorbanceat 650 nm or 675 nmFigure 10 shows a plot of the hemoglobinconcentration as determined using the absorbance assay described hereinand hemoglobin concentration determined using the Sysmex XS-1000iautomated hematology system. As shown in FIG. 10, there is a stronglinear relationship between the hemoglobin concentrations determinedusing the subject methods as compared to the hematology analyzer. Thisshows that the subject methods are suitable for providing clinicallyaccurate concentrations of hemoglobin in whole blood (venipuncture orfingerstick).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

1-50. (canceled)
 50. A system for assaying a sample for an analyte, thesystem comprising: a broad spectrum light source; a slit projectionmodule coupled to the broad spectrum light source, wherein the slitprojection module comprises: a slit that narrows a beam of light fromthe broad spectrum light source to a width equal to the width of theslit; and a focusing lens that focuses light from the slit; an objectivelens which focuses light transmitted from a sample; a diffractiongrating; and a detector for detecting one or more predeterminedwavelengths of the transmitted light.
 51. The system according to claim50, wherein the broad spectrum light source comprises a visible lightsource and a near infrared light source.
 52. The system according toclaim 51, wherein the broad spectrum light source comprises anirradiation profile having emission peaks at about 450 nm, about 550 nmand about 830 nm.
 53. The system according to claim 50, wherein the slitprojection module comprises: a slit configured to narrow a beam of lightfrom the broad spectrum light source; and a focusing lens coupled to theslit for focusing light which passes through the slit.
 54. The systemaccording to claim 53, wherein the slit has a width of from about 75 μmto 125 μm. 55-58. (canceled)
 59. The system according to claim 53,wherein the slit projection module is configured to project a light beamin the shape of a slit having a length of from about 2.5 mm to about 3.5mm.
 60. (canceled)
 61. The system according to claim 53, wherein theslit projection module is configured to project a light beam in theshape of a slit having a width of from about 25 μm to about 75 μm.62-63. (canceled)
 64. The system according to claim 53, wherein thefocusing lens coupled to the slit comprises a de-magnifying lens. 65.The system according to claim 64, wherein the focusing lens is a doubletachromatic lens.
 66. The system according to claim 64, wherein thefocusing lens coupled to the slit has a magnification ratio of fromabout 0.5 to about 0.75.
 67. (canceled)
 68. The system according toclaim 50, wherein the slit projection module is configured to move alongthe length of a microfluidic chamber. 69-70. (canceled)
 71. The systemaccording to claim 50, wherein the system is configured to move amicrofluidic chamber relative to the slit projection module. 72-73.(canceled)
 74. The system according to claim 50, wherein the objectivelens has a magnification ratio of from 1.5 to 2.5.
 75. (canceled) 76.The system according to claim 50, wherein the objective lens is adoublet achromatic lens.
 77. The system according to claim 50, whereinthe system further comprises a diffraction grating configured tospatially separate light into separate wavelengths.
 78. (canceled) 79.The system according to claim 50, wherein the slit projection module,objective lens and diffraction grating provide a spatial separationresolution of 5 nm or less.
 80. (canceled)
 81. The system according toclaim 50, wherein the detector is a charged coupled device. 82-87.(canceled)
 88. A system comprising: a broad spectrum light source; aslit projection module coupled to the broad spectrum light source,wherein the slit projection module comprises: a slit that narrows a beamof light from the broad spectrum light source to a width equal to thewidth of the slit; and a focusing lens that focuses light from the slit;a cartridge holder configured to receive a microfluidic device having acapillary channel sample chamber; and a detector for detecting one ormore predetermined wavelengths of the transmitted light; and amicrofluidic device configured to perform an assay of a liquid samplepositioned in the cartridge holder, the device comprising: a sampleapplication site in fluid communication with an inlet into a capillarychannel sample chamber; a capillary channel sample chamber; and areagent mixing chamber positioned between the sample application siteand the capillary channel sample chamber. 89-135. (canceled)
 136. Amethod comprising: providing a system for assaying a sample for ananalyte, the system comprising: a broad spectrum light source; a slitprojection module coupled to the broad spectrum light source, whereinthe slit projection module comprises: a slit that narrows a beam oflight from the broad spectrum light source to a width equal to the widthof the slit; and a focusing lens that focuses light from the slit; acartridge holder configured to receive a microfluidic device having acapillary channel sample chamber; and a detector for detecting one ormore predetermined wavelengths of the transmitted light; positioning themicrofluidic device into the cartridge holder; illuminating a sample ina sample chamber with a light source through a slit projection module toprovide a slit-shaped beam on the sample chamber; detecting lighttransmitted through the sample; and calculating absorbance of thedetected light at one or more wavelengths to assay the sample for theanalyte in the sample. 137-173. (canceled)